Peptides that bind to the erythropoietin receptor

ABSTRACT

The present invention relates to peptide compounds that are agonists of the erythropoietin receptor (EPO-R). The invention also relates to therapeutic methods using such peptide compounds to treat disorders associated with insufficient or defective red blood cell production. Pharmaceutical compositions, which comprise the peptide compounds of the invention, are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.10/555,868, filed Nov. 2, 2005, which is a 371 National PhaseApplication of International Application No. PCT/US2004/014886, filedMay 12, 2004, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application No. 60/470,245, filed May 12, 2003. TheInternational Application was published in English under PCT Article21(2) as International Publication No. WO 2004/11016 on Nov. 25, 2004.The entire contents of each of these applications is incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to peptide compounds that are agonists ofthe erythropoietin receptor (EPO-R). The invention also relates totherapeutic methods using such peptide compounds to treat disordersassociated with insufficient or defective red blood cell production.Pharmaceutical compositions, which comprise the peptide compounds of theinvention, are also provided.

BACKGROUND OF THE INVENTION

Erythropoietin (EPO) is a glycoprotein hormone of 165 amino acids, witha molecular weight of about 34 kilodaltons (kD) and preferredglycosylation sites on amino-acid positions 24, 38, 83, and 126. It isinitially produced as a precursor protein with a signal peptide of 23amino acids. EPO can occur in three forms: α, β, and asialo. The α and βforms differ slightly in their carbohydrate components, but have thesame potency, biological activity, and molecular weight. The asialo formis an α or β form with the terminal carbohydrate (sialic acid) removed.The DNA sequences encoding EPO have been reported [U.S. Pat. No.4,703,008 to Lin].

EPO stimulates mitotic division and differentiation of erythrocyteprecursor cells, and thus ensures the production of erythrocytes. It isproduced in the kidney when hypoxic conditions prevail. DuringEPO-induced differentiation of erythrocyte precursor cells, globinsynthesis is induced; heme complex synthesis is stimulated; and thenumber of ferritin receptors increases. These changes allow the cell totake on more iron and synthesize functional hemoglobin, which in matureerythrocytes binds oxygen. Thus, erythrocytes and their hemoglobin playa key role in supplying the body with oxygen. These changes areinitiated by the interaction of EPO with an appropriate receptor on thecell surface of the erythrocyte precursor cells [See, e.g., Graber andKrantz (1978) Ann. Rev. Med. 29.51-66].

EPO is present in very low concentrations in plasma when the body is ina healthy state wherein tissues receive sufficient oxygenation from theexisting number of erythrocytes. This normal low concentration issufficient to stimulate replacement of red blood cells which are lostnormally through aging.

The amount of EPO in the circulation is increased under conditions ofhypoxia when oxygen transport by blood cells in the circulation isreduced. Hypoxia may be caused, for example, by substantial blood lossthrough hemorrhage, destruction of red blood cells by over-exposure toradiation, reduction in oxygen intake due to high altitude or prolongedunconsciousness, or various forms of anemia. In response to such hypoxicstress, elevated EPO levels increase red blood cell production bystimulating the proliferation of erythroid progenitor cells. When thenumber of red blood cells in circulation is greater than needed fornormal tissue oxygen requirements, EPO levels in circulation aredecreased.

Because EPO is essential in the process of red blood cell formation,this hormone has potentially useful applications in both the diagnosisand the treatment of blood disorders characterized by low or defectivered blood cell production. Recent studies have provided a basis for theprojection of EPO therapy efficacy for a variety of disease states,disorders, and states of hematologic irregularity, including:beta-thalassemia [see, Vedovato, et al. (1984) Acta. Haematol.71:211-213]; cystic fibrosis [see, Vichinsky, et al. (1984) J. Pediatric105:15-21]; pregnancy and menstrual disorders [see, Cotes, et al. (193)Brit. J. Ostet. Gyneacol. 90:304-311]; early anemia of prematurity [see,Haga, et al. (1983) Acta Pediatr. Scand. 72; 827-831]; spinal cordinjury [see, Claus-Walker, et al. (1984) Arch. Phys. Med. Rehabil.65:370-374]; space flight [see, Dunn, et al. (1984) Eur. J. Appl.Physiol. 52:178-182]; acute blood loss [see, Miller, et al., (1982)Brit. J. Haematol. 52:545-590]; aging [see, Udupa, et al. (1984) J. Lab.Clin. Med. 103:574-580 and 581-588 and Lipschitz, et al. (1983) Blood63:502-509]; various neoplastic disease states accompanied by abnormalerythropoiesis [see, Dainiak, et al. (1983) Cancer 5:1101-1106 andSchwartz, et al. (1983) Otolaryngol. 109:269-272]; and renalinsufficiency [see, Eschbach. et al. (1987) N. Eng. J. Med. 316:73-78].

Purified, homogeneous EPO has been characterized [U.S. Pat. No.4,677,195 to Hewick]. A DNA sequence encoding EPO was purified, cloned,and expressed to produce recombinant polypeptides with the samebiochemical and immunological properties as natural EPO. A recombinantEPO molecule with oligosaccharides identical to those on natural EPO hasalso been produced [See, Sasaki, et al. (1987) J. Biol. Chem.262:12059-12076].

The biological effect of EPO appears to be mediated, in part, throughinteraction with a cell membrane bound receptor. Initial studies, usingimmature erythroid cells isolated from mouse spleen, suggested that theEPO-binding cell surface proteins comprise two polypeptides havingapproximate molecular weights of 85,000 Daltons and 100,000 Daltons,respectively [Sawyer, et al. (1987) Proc. Natl. Acad. Sci. USA84:3690-3694]. The number of EPO-binding sites was calculated to averagefrom 800 to 1000 per cell surface. Of these binding sites, approximately300 bound EPO with a K_(d) of approximately 90 μM (picomolar), while theremaining bound EPO with a reduced affinity of approximately 570 μM[Sawyer, et al. (1987) J. Biol. Chem. 262:5554-5562]. An independentstudy suggested that EPO-responsive splenic erythroblasts, prepared frommice injected with the anemic strain (FVA) of the Friend leukemia virus,possess a total of approximately 400 high and low affinity EPO bindingsites with K_(d) values of approximately 100 μM and 800 μM, respectively[Landschulz, et al. (1989) Blood 73:1476-1486].

Subsequent work indicated that the two forms of EPO receptor (EPO-R)were encoded by a single gene. This gene has been cloned [See, e.g.,Jones, et al. (1990) Blood 76, 31-35; Noguchi, et al. (1991) Blood78:2548-2556; Maouche, et al. (1991) Blood 78:2557-2563]. For example,the DNA sequences and encoded peptide sequences for murine and humanEPO-R proteins are described in PCT Pub. No. WO 90/08822 to D'Andrea, etal. Current models suggest that binding of EPO to EPO-R results in thedimerization and activation of two EPO-R molecules, which results insubsequent steps of signal transduction [See, e.g., Watowich, et al.(1992) Proc. Natl. Acad. Sci. USA 89:2140-2144].

The availability of cloned genes for EPO-R facilitates the search foragonists and antagonists of this important receptor. The availability ofthe recombinant receptor protein allows the study of receptor-ligandinteraction in a variety of random and semi-random peptide diversitygeneration systems. These systems include the “peptides on plasmids”system [described in U.S. Pat. No. 6,270,170]; the “peptides on phage”system [described in U.S. Pat. No. 5,432,018 and Cwirla, et al. (1990)Proc. Natl. Acad. Sci. USA 87:6378-6382]; the “encoded syntheticlibrary” (ESL) system [described in U.S. patent application Ser. No.946,239, filed Sep. 16, 1992]; and the “very large scale immobilizedpolymer synthesis” system [described in U.S. Pat. No. 5,143,854; PCTPub. No. 90/15070; Fodor, et al. (1991) Science 251:767-773; Dower andFodor (1991) Ann. Rep. Med. Chem. 26:271-180; and U.S. Pat. No.5,424,186].

Peptides that interact to a least some extent with EPO-R have beenidentified and are described, for example in U.S. Pat. Nos. 5,773,569;5,830,851; and 5,986,047 to Wrighton, et al.; PCT Pub. No. WO 96/40749to Wrighton, et al.; U.S. Pat. No. 5,767,078 and PCT Pub. No. 96/40772to Johnson and Zivin; PCT Pub. No. WO 01/38342 to Balu; and WO 01/91780to Smith-Swintosky, et al. In particular, a group of peptides containinga peptide motif has been identified, members of which bind to EPO-R andstimulate EPO-dependent cell proliferation. Yet, peptides identified todate that contain the motif stimulate EPO-dependent cell proliferationin vitro with EC50 values between about 20 nanomolar (DM) and 250 nM.Thus, peptide concentrations of 20 nM to 250 nM are required tostimulate 50% of the maximal cell proliferation stimulated by EPO.

Given the immense potential of EPO-R agonists, both for studies of theimportant biological activities mediated by this receptor and fortreatment of disease, there remains a need for the identification ofpeptide EPO-R agonists of enhanced potency and activity. The presentinvention provides such compounds.

The citation and/or discussion of cited references in this section andthroughout the specification is provided merely to clarify thedescription of the present invention and is not an admission that anysuch reference is “prior art” to the present invention.

SUMMARY OF THE INVENTION

The present invention provides EPO-R agonist peptides of dramaticallyenhanced potency and activity. These agonists include monomeric peptideagonists of 17 to about 40 amino acids in length that comprise the coreamino acid sequence LYACHX₀GPITX₁VCQPLR (SEQ ID NO: 1), where each aminoacid is indicated by standard one letter abbreviation, X₀ is methionine(M) or homoserine methylether (Hsm), and X₁ is tryptophan (W),1-naphthylalanine (1-nal), or 2-naphthylalanine (2-nal); as well asdimeric peptide agonists that comprise two peptide monomers, whereineach peptide monomer is of 17 to about 40 amino acids in length andcomprises the core amino acid sequence LYACHX₀GPITX₁VCQPLR (SEQ IDNO:1), where each amino acid is indicated by standard one letterabbreviation, X₀ is methionine (M) or homoserine methylether (Hsm), andX₁ is tryptophan (W), 1-naphthylalanine (1-nal), or 2-naphthylalanine(2-nal). The potency of these novel peptide agonists may be furtherenhanced by one or more modifications, including: acetylation,intramolecular disulfide bond formation, and covalent attachment of oneor more polyethylene glycol (PEG) moieties. The invention furtherprovides pharmaceutical compositions comprised of such peptide agonists,and methods to treat various medical conditions using such peptideagonists.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn orN; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Gluor E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg orR; and Glycine is Gly or G. The unconventional amino acids in peptidesare abbreviated as follows: 1-naphthylalanine is 1-nal or Np;2-naphthylalanine is 2-nal; N-methylglycine (also known as sarcosine) isMeG or Sc; homoserine methylether is Hsm; and acetylated glycine(N-acetylglycine) is AcG.

As used herein, the term “polypeptide” or “protein” refers to a polymerof amino acid monomers that are alpha amino acids joined togetherthrough amide bonds. Polypeptides are therefore at least two amino acidresidues in length, and are usually longer. Generally, the term“peptide” refers to a polypeptide that is only a few amino acid residuesin length. The novel EPO-R agonist peptides of the present invention arepreferably no more than about 50 amino acid residues in length. They aremore preferably from about 17 to about 40 amino acid residues in length.A polypeptide, in contrast with a peptide, may comprise any number ofamino acid residues. Hence, the term polypeptide included peptides aswell as longer sequences of amino acids.

As used herein, the phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that are “generally regarded assafe”, e.g., that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water or aqueous solution saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E.W. Martin.

As used herein the term “agonist” refers to a biologically active ligandwhich binds to its complementary biologically active receptor andactivates the latter either to cause a biological response in thereceptor, or to enhance preexisting biological activity of the receptor.

Novel Peptides that are EPO-R Agonists

The present invention relates to peptides that are agonists of the EPO-Rand show dramatically enhanced potency and activity. These peptideagonists are preferably of 17 to about 40 amino acids in length andcomprise the core amino acid sequence LYACHX₀GPITX₁VCQPLR (SEQ ID NO:1), where each amino acid is indicated by standard one letterabbreviation, X₀ is methionine (M) or homoserine methylether (Hsm), andX₁ is tryptophan (W), 1-naphthylalanine (1-nal), or 2-naphthylalanine(2-nal).

The peptides of this invention may be monomers, dimers, or othermultimers. The peptide multimers of the invention may be trimers,tetramers, pentamers, or other higher order structures. Moreover, suchdimers and other multimers may be heterodimers or heteromultimers. Thepeptide monomers of the present invention may be degradation products(e.g., oxidation products of methionine or deamidated glutamine,arganine, and C-terminus amide). Such degradation products may be usedin and are therefore considered part of the present invention. Inpreferred embodiments, the heteromultimers of the invention comprisemultiple peptides that are all EPO-R agonist peptides. In highlypreferred embodiments, the multimers of the invention are homomultimers:i.e., they comprise multiple EPO-R agonist peptides of the same aminoacid sequence.

Accordingly, the present invention also relates to dimeric peptideagonists of EPO-R, which show dramatically enhanced potency andactivity. In preferred embodiments, the dimers of the invention comprisetwo peptides that are both EPO-R agonist peptides. These preferreddimeric peptide agonists comprise two peptide monomers, wherein eachpeptide monomer is of 17 to about 40 amino acids in length and comprisesthe core amino acid sequence LYACHX₀GPITX₁VCQPLR (SEQ ID NO: 1), whereeach amino acid is indicated by standard one letter abbreviation, X₀ ismethionine (M) or homoserine methylether (Hsm), and X₁ is tryptophan(W), 1-naphthylalanine (1-nal), or 2-naphthylalanine (2-nal). Inparticularly preferred embodiments, the dimers of the invention comprisetwo EPO-R agonist peptides of the same amino acid sequence.

According to some embodiments of the invention, two or more, andpreferably from two to six amino acid residues, independently selectedfrom any of the 20 genetically encoded L-amino acids or thestereoisomeric D-amino acids, will be coupled to either or both ends ofthe core sequence described above. For example, the sequence GG willoften be appended to either terminus or both termini of the coresequence.

Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α,α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for compounds of the present invention.Examples of unconventional amino acids include, but are not limited to:β-alanine, 3-pyridylalanine, 4-hydroxyproline, O-phosphoserine,N-methylglycine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, nor-leucine, and other similar amino acids and iminoacids.

Other modifications are also possible, including modification of theamino terminus, modification of the carboxy terminus, replacement of oneor more of the naturally occurring genetically encoded amino acids withan unconventional amino acid, modification of the side chain of one ormore amino acid residues, peptide phosphorylation, and the like. Apreferred amino terminal modification is acetylation (e.g., with aceticacid or a halogen substituted acetic acid). In preferred embodiments anN-terminal glycine is acetylated to N-acetylglycine (AcG). In preferredembodiments, a the C-terminal glycine is N-methylglycine (MeG, alsoknown as sarcosine).

Preferred peptide monomers of the present invention include, but are notlimited to:

(SEQ ID NO: 2) LYACHMGPITWVCQPLR; (SEQ ID NO: 3)LYACHMGPIT(1-nal)VCQPLR; (SEQ ID NO: 4) LYACHMGPIT(2-nal)VCQPLR;(SEQ ID NO: 5) GGLYACHMGPITWVCQPLRG; (SEQ ID NO: 6)GGLYACHMGPIT(1-nal)VCQPLRG; (SEQ ID NO: 7) GGLYACHMGPIT(2-nal)VCQPLRG;(SEQ ID NO: 8) (AcG)GLYACHMGPITWVCQPLRG; (SEQ ID NO: 9)(AcG)GLYACHMGPIT(1-nal)VCQPLRG; (SEQ ID NO: 10)(AcG)GLYACHMGPIT(2-nal)VCQPLRG; (SEQ ID NO: 11)GGLYACHMGPITWVCQPLR(MeG); (SEQ ID NO: 12)GGLYACHMGPIT(1-nal)VCQPLR(MeG); (SEQ ID NO: 13)GGLYACHMGPIT(2-nal)VCQPLR(MeG); (SEQ ID NO: 14)(AcG)GLYACHMGPITWVCQPLRG(MeG); (SEQ ID NO: 15)(AcG)GLYACHMGPIT(1-nal)VCQPLRG(MeG); (SEQ ID NO: 16)(AcG)GLYACHMGPIT(2-nal)VCQPLRG(MeG); (SEQ ID NO: 17)LYACH(Hsm)GPITWVCQPLR; (SEQ ID NO: 18) LYACH(Hsm)GPIT(1-nal)VCQPLR;(SEQ ID NO: 19) LYACH(Hsm)GPIT(2-nal)VCQPLR; (SEQ ID NO: 20)GGLYACH(Hsm)GPITWVCQPLRG; (SEQ ID NO: 21)GGLYACH(Hsm)GPIT(1-nal)VCQPLRG; (SEQ ID NO: 22)GGLYACH(Hsm)GPIT(2-nal)VCQPLRG; (SEQ ID NO: 23)(AcG)GLYACH(Hsm)GPITWVCQPLRG; (SEQ ID NO: 24)(AcG)GLYACH(Hsm)GPIT(1-nal)VCQPLRG; (SEQ ID NO: 25)(AcG)GLYACH(Hsm)GPIT(2-nal)VCQPLRG; (SEQ ID NO: 26)GGLYACH(Hsm)GPITWVCQPLR(MeG); (SEQ ID NO: 27)GGLYACH(Hsm)GPIT(1-nal)VCQPLR(MeG); (SEQ ID NO: 28)GGLYACH(Hsm)GPIT(2-nal)VCQPLR(MeG); (SEQ ID NO: 29)(AcG)GLYACH(Hsm)GPITWVCQPLRG(MeG); (SEQ ID NO: 30)(AcG)GLYACH(Hsm)GPIT(1-nal)VCQPLRG(MeG;) and. (SEQ ID NO: 31)(AcG)GLYACH(Hsm)GPIT(2-nal)VCQPLRG(MeG).

In preferred embodiments, the peptide monomers of the invention containan intramolecular disulfide bond between the two cysteine residues ofthe core sequence. Such monomers may be represented schematically asfollows:

The present invention also provides conjugates of these peptidemonomers. Thus, according to a preferred embodiment, the monomericpeptides of the present invention are dimerized or oligomerized, therebyenhancing EPO-R agonist activity.

In one embodiment, the peptide monomers of the invention may beoligomerized using the biotin/streptavidin system. Biotinylated analogsof peptide monomers may be synthesized by standard techniques. Forexample, the peptide monomers may be C-terminally biotinylated. Thesebiotinylated monomers are then oligomerized by incubation withstreptavidin [e.g., at a 4:1 molar ratio at room temperature inphosphate buffered saline (PBS) or HEPES-buffered RPMI medium(Invitrogen) for 1 hour]. In a variation of this embodiment,biotinylated peptide monomers may be oligomerized by incubation with anyone of a number of commercially available anti-biotin antibodies [e.g.,goat anti-biotin IgG from Kirkegaard & Perry Laboratories, Inc.(Washington, D.C.)].

In preferred embodiments, the peptide monomers of the invention aredimerized by covalent attachment to at least one linker moiety. Thelinker (L_(K)) moiety is preferably, although not necessarily, a C₁₋₁₂linking moiety optionally terminated with one or two —NH— linkages andoptionally substituted at one or more available carbon atoms with alower alkyl substituent. Preferably the linker L_(K) comprises —NH—R—NH—wherein R is a lower (C₁₋₆) alkylene substituted with a functional groupsuch as a carboxyl group or an amino group that enables binding toanother molecular moiety (e.g., as may be present on the surface of asolid support). Most preferably the linker is a lysine residue or alysine amide (a lysine residue wherein the carboxyl group has beenconverted to an amide moiety —CONH₂). In preferred embodiments, thelinker bridges the C-termini of two peptide monomers, by simultaneousattachment to the C-terminal amino acid of each monomer.

For example, when the C-terminal linker L_(K) is a lysine amide thedimer may be illustrated structurally as shown in Formula I, andsummarized as shown in Formula II:

In Formula I and Formula II, N² represents the nitrogen atom of lysine's6-amino group and N′ represents the nitrogen atom of lysine's α-aminogroup. The dimeric structure can be written as [peptide]₂Lys-amide todenote a peptide bound to both the α and ε amino groups of lysine, or[Ac-peptide]₂Lys-amide to denote an N-terminally acetylated peptidebound to both the α and ε amino groups of lysine, or [Ac-peptide,disulfide]₂Lys-amide to denote an N-terminally acetylated peptide boundto both the α and ε amino groups of lysine with each peptide containingan intramolecular disulfide loop, or [Ac-peptide,disulfide]₂Lys-spacer-PEG to denote an N-terminally acetylated peptidebound to both the α and ε amino groups of lysine with each peptidecontaining an intramolecular disulfide loop and a spacer moleculeforming a covalent linkage between the C-terminus of lysine and a PEGmoiety, or [Ac-peptide-Lys*-NH₂]₂-Iminodiacetic-N-(Boc-β3Ala) to denotea homodimer of an N-terminally acetylated peptide bearing a C-terminallysineamide residue where the ε amine of lysine is bound to each of thetwo carboxyl groups of iminodiacetic acid and where Boc-beta-alanine iscovalently bound to the nitrogen atom of iminodiacetic acid via an amidebond.

In an additional embodiment, polyethylene glycol (PEG) may serve as thelinker L_(K) that dimerizes two peptide monomers: for example, a singlePEG moiety may be simultaneously attached to the N-termini of bothpeptide chains of a peptide dimer.

In yet another additional embodiment, the linker (L_(K)) moiety ispreferably, but not necessarily, a molecule containing two carboxylicacids and optionally substituted at one or more available atoms with anadditional functional group such as an amine capable of being bound toone or more PEG molecules. Such a molecule can be depicted as:

—CO—(CH₂)_(n)—X—(CH₂)_(m)—CO—

where n is an integer from 0 to 10, m is an integer from 1 to 10, X isselected from O, S, N(CH₂)_(p)NR₁, NCO(CH₂)_(p)NR₁, and CHNR₁, R₁ isselected from H, Boc, Cbz, etc., and p is an integer from 1 to 10.

In preferred embodiments, one amino group of each of the peptides forman amide bond with the linker L_(K). In particularly preferredembodiments, the amino group of the peptide bound to the linker L_(K) isthe epsilon amine of a lysine residue or the alpha amine of theN-terminal residue, or an amino group of the optional spacer molecule.In particularly preferred embodiments, both n and m are one, X isNCO(CH₂)_(p)NR₁, p is two, and R₁ is Boc. A dimeric EPO peptidecontaining such a preferred linker may be structurally illustrated asshown in Formula III.

Optionally, the Boc group can be removed to liberate a reactive aminegroup capable of forming a covalent bond with a suitably activated watersoluble polymer species, for example, a PEG species such asmPEG-para-nitrophenylcarbonate (mPEG-NPC), mPEG-succinimidyl propionate(mPEG-SPA), and N-hydroxysuccinimide-PEG (NHS-PEG) (see, e.g., U.S. Pat.No. 5,672,662). A dimeric EPO peptide containing such a preferred linkermay be structurally illustrated as shown in Formula IV.

Generally, although not necessarily, peptide dimers will also containone or more intramolecular disulfide bonds between cysteine residues ofthe peptide monomers. Preferably, the two monomers contain at least oneintramolecular disulfide bond. Most preferably, both monomers of apeptide dimer contain an intramolecular disulfide bond, such that eachmonomer contains a cyclic group.

A peptide monomer or dimer may further comprise one or more spacermoieties. Such spacer moieties may be attached to a peptide monomer orto a peptide dimer. Preferably, such spacer moieties are attached to thelinker L_(K) moiety that connects the monomers of a peptide dimer. Forexample, such spacer moieties may be attached to a peptide dimer via thecarbonyl carbon of a lysine linker, or via the nitrogen atom of animinodiacetic acid linker. For example, such a spacer may connect thelinker of a peptide dimer to an attached water soluble polymer moiety ora protecting group. In another example, such a spacer may connect apeptide monomer to an attached water soluble polymer moiety.

In one embodiment, the spacer moiety is a C₁₋₁₂ linking moietyoptionally terminated with —NH-linkages or carboxyl (—COOH) groups, andoptionally substituted at one or more available carbon atoms with alower alkyl substituent. In one embodiment, the spacer is R—COOH whereinR is a lower (C₁₋₆) alkylene optionally substituted with a functionalgroup such as a carboxyl group or an amino group that enables binding toanother molecular moiety. For example, the spacer may be a glycine (G)residue, or an amino hexanoic acid. In preferred embodiments the aminohexanoic acid is 6-amino hexanoic acid (Ahx). For example, where thespacer 6-amino hexanoic acid (Ahx) is bound to the N-terminus of apeptide, the peptide terminal amine group may be linked to the carboxylgroup of Ahx via a standard amide coupling. In another example, whereAhx is bound to the C-terminus of a peptide, the amine of Ahx may belinked to the carboxyl group of the terminal amino acid via a standardamide coupling. The structure of such a peptide may be depicted as shownin Formula V, and summarized as shown in Formula VI.

In other embodiments, the spacer is —NH—R—NH— wherein R is a lower(C₁₋₆) alkylene substituted with a functional group such as a carboxylgroup or an amino group that enables binding to another molecularmoiety. For example, the spacer may be a lysine (K) residue or a lysineamide (K—NH₂, a lysine residue wherein the carboxyl group has beenconverted to an amide moiety —CONH₂).

In preferred embodiments, the spacer moiety has the following structure:

—NH—(CH₂)_(α)[O—(CH₂)_(β)]_(γ)—O_(δ)—(CH₂)_(ε)—Y—

where α, β, γ, δ, and ε are each integers whose values are independentlyselected. In preferred embodiments, α, β, and ε are each integers whosevalues are independently selected from one to about six, δ is zero orone, γ is an integer selected from zero to about ten, except that when γis greater than one, β is two, and Y is selected from NH or CO. Inparticularly preferred embodiments α, β, and ε are each equal to two,both γ and δ are equal to 1, and Y is NH. For example, a peptide dimercontaining such a spacer is illustrated schematically in Formula VII,where the linker is a lysine and the spacer joins the linker to a Bocprotecting group.

In another particularly preferred embodiment γ and δ are zero, α and εtogether equal five, and Y is CO.

In particularly preferred embodiments, the linker plus spacer moiety hasthe structure shown in Formula VIII or Formula IX.

The peptide monomers, dimers, or multimers of the invention may furthercomprise one or more water soluble polymer moieties. Preferably, thesepolymers are covalently attached to the peptide compounds of theinvention. Preferably, for therapeutic use of the end-productpreparation, the polymer will be pharmaceutically acceptable. Oneskilled in the art will be able to select the desired polymer based onsuch considerations as whether the polymer-peptide conjugate will beused therapeutically, and if so, the desired dosage, circulation time,resistance to proteolysis, and other considerations. The water solublepolymer may be, for example, polyethylene glycol (PEG), copolymers ofethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,polypropylene oxide/ethylene oxide copolymers, and polyoxyethylatedpolyols. A preferred water soluble polymer is PEG.

The polymer may be of any molecular weight, and may be branched orunbranched. A preferred PEG for use in the present invention compriseslinear, unbranched PEG having a molecular weight that is greater than 10kilodaltons (kD) and is more preferably between about 20 and 60 kD inmolecular weight. Still more preferably, the linear unbranched PEGmoiety should have a molecular weight of between about 20 and 40 kD,with 20 kD PEG being particularly preferred. It is understood that in agiven preparation of PEG, the molecular weights will typically varyamong individual molecules. Some molecules will weight more, and someless, than the stated molecular weight. Such variation is generallyreflect by use of the word “about” to describe molecular weights of thePEG molecules.

The number of polymer molecules attached may vary; for example, one,two, three, or more water soluble polymers may be attached to an EPO-Ragonist peptide of the invention. The multiple attached polymers may bethe same or different chemical moieties (e.g., PEGs of differentmolecular weight). Thus, in a preferred embodiment the inventioncontemplates EPO-R agonist peptides having two or more PEG moieitiesattached thereto. Preferably, both of the PEG moietieis are linear,unbranched PEG each preferably having a molecular weight of betweenabout 10 and about 601(1). More preferably, each linear unbranched PEGmoiety has a molecular weight that is between about 20 and 40 kD, andstill more preferably between about 20 and 30 kD with a molecular weightof about 20 kD for each linear PEG moiety being particularly preferred.However, other molecular weights for PEG are also contemplated in suchembodiments. For example, the invention contemplates and encompassesEPO-R agonist peptides having two or more linear unbranched PEG moietiesattached thereto, at least one or both of which has a molecular weightbetween about 20 and 40 kD or between about 20 and 30 kD. In otherembodiments the invention contemplates and encompasses EPO-R agonistpeptides having two or more linear unbranched PEG moieties attachedthereto, at least one of which has a molecular weight between about 40and 60 kD.

In one embodiment, PEG may serve as a linker that dimerizes two peptidemonomers. In one embodiment, PEG is attached to at least one terminus(N-terminus or C-terminus) of a peptide monomer or dimer. In anotherembodiment, PEG is attached to a spacer moiety of a peptide monomer ordimer. In a preferred embodiment PEG is attached to the linker moiety ofa peptide dimer. In a highly preferred embodiment, PEG is attached to aspacer moiety, where said spacer moiety is attached to the linker L_(K)moiety that connects the monomers of a peptide dimer. In particularlypreferred embodiments, PEG is attached to a spacer moiety, where saidspacer moiety is attached to a peptide dimer via the carbonyl carbon ofa lysine linker, or the amide nitrogen of a lysine amide linker.

Preferred peptide dimers of the present invention include, but are notlimited to:

Compound designation Peptide dimer   AF33065

AF34602

AF34395

AF34601

AF32579

AF33068

AF33131

AF34351

AF34350

AF34753

AF34757

AF35062

AF35218

AF35462

AF35464

AF33197

AF34994

AF35083

AF35525

AF35526

AF35563

AF35575

AF35592

AF35593

AF35594

AF35219

AF32876

AF32881

AF35179

AF35180

AF35463

AF35090

AF35148

AF35149

AF35168

AF35361

AF35595

AF35564

Still other peptides of the present invention, including peptidemonomers and dimers, are as follows:

SEQ ID PEG NO: size 34 Ac G G L Y A C H M G P I T 1 Nal 35 [ Ac G G L YA C H M G P I T 1 Nal 36 [ Ac G G L Y A C H M G P I T 1 Nal 37 40 [ Ac GG L Y A C H M G P I T 1 Nal KDa 38 39 [ Ac G G L Y A C H M(O2 G P I T 1Nal 40 40 [ Ac G G L Y A C H M(O2 G P I T 1 Nal KDa 41 [ Ac G G L Y A CH M(O) G P I T 1 Nal 42 [ Ac G G L Y A C H M(O) G P I T 1 Nal 43 [ Ac GG L Y A C H MXX G P I T 1 Nal 44 [ Ac G G L Y A C H MXX G P I T 1 Nal 45[ Ac G G L Y A C H MXX G P I T 1 Nal 46 [

G L Y A C H M G P I T 1 Nal 47 [

G G L Y A C H M G P I T 1 Nal 48 [ Ac G G L Y A C H M G P I T 1 Nal 49 [Ac G G L Y A C H M(O) G P I T 1 Nal 50 [ Ac G G L Y A C H M G P O T 1Nal 51 [ Ac S R T R Y R C E M G P L T W 52 [ Ac L T R L Y S C H M G P ST W 53 [ Ac R G Q L Y A C H F G P V T W 54 [ Ac S G I L Y E C H M G P LT W 55 [ Ac L G R R Y S C H F G A L T W 56 [ Ac G S R T Y S C Q L G P VD W 57 Ac A R G R Y Q C Q F G P L T W 58 Ac V T R M Y R C R M G P L T W59 Ac R P S L Y E C H L G P L T W 60 Ac R G H M Y S C Q L G P V T W 61Ac I T P T Y H C R F G P Q T W 62 Ac G N R M Y Q C H M G P L T W 63 [ AcA R G R Y Q C Q F G P L T W 64 [ Ac V T R M Y R C R M G P L T W 65 [ AcR P S L Y E C H L G P L T W 66 [ Ac R G H M Y S C Q L G P V T W 67 [ AcI T P T Y H C R F G P Q T W 68 [ Ac G N R M Y Q C H M G P L T W 69 [ AcR N H L Y G C R M G P L T W 70 [ Ac P D L A Y S C R M G P L T W 71 [ AcL G R R Y S C H F G P L T W 72 [ Ac L L R G Y E C Y M G P L T W 73 [ AcM R T R Y R C Y M G P L T W 74 [ Ac H L G R Y D C S F G P Q T W 75 [ AcI R G R N R C R F G P Q T W 76 Ac Q R R H V F L S D G A A Y V G L W 77 [Ac V L P L Y R C R M G R E T W 78 [ Ac P G N S Y R C H M G P L T W 79 AcR N H L Y G C R M G P L T W 80 Ac P D L A Y S C R M G P L T W 81 Ac L GR R Y S C H F G P L T W 82 Ac L L R G Y E C Y M G P L T W 83 Ac M R T RY R C Y M G P L T W 84 Ac H L G R Y D C S F G P Q T W 85 Ac I R G R N RC R F G P Q T W 86 Ac R P R P Y S C T M G P R T W 87 Ac V L P L Y R C RM G R E T W 88 Ac P G N S Y R C M G P L T W V 89 40 [ Ac G G L Y A C HM(x) G P I T 1 Nal KDa 90 40 [ Ac G G L Y A C H M(x) G P I T 1 Nal KDa91 [ Ac G G I Y A C H M G P I T 1 Nal 92 [ Ac G G L Y A C H m G P I T 1Nal 93 [ Ac G G L Y A c H M G P I T 1 Nal 94 [ Ac G G L Y A c H M G P IT 1 Nal 95 [ Ac G G L Y A C H M G P I T 1 Nal 96 [ Ac G G L Y A C H M GP I T 1 Nal 97 [ Ac E Y L C R M G P I T W 98 [ Ac T Y S C H F G P L T W99 [ Ac D Y H C R M G P L T W 100 [ Ac L Y E C R M G P M T W 101 [ Ac LY L C R M G P V T W 102 [ Ac D Y N C R F G P L T W 103 [ Ac S Y L C R RG P T T W 104 [ Ac E Y S C R M G P M T W 105 [ Ac L Y L C R F G P V T W106 [ Ac I Y R C L M G P L T W 107 [ Ac G G L Y A C H M G P I T 1 Nal108 40 [

G G L Y A C H M G P I T 1 Nal KDa 109 40 [ G G L Y A C H M G P I T 1 NalKDa 110 [ Ac G G L Y A C H M G p I T 1 Nal 111 [ Ac G G L Y A C h M G PI T 1 Nal 112 [ Ac G G L Y a C H M G P I T 1 Nal 113 [ Ac G G L Y A CxxH M G P I T 1 Nal 114 [ Ac G G L Y A C (Acm) H M G P I T 1 Nal 115 40 [

G L Y A C H M G P I T 1 Nal KDa 116 40 [ Ac G G L Y A C H M G P I T 1Nal KDa 117 [ Ac G G L Y A CSH H M G P I T 1 Nal 118 [ Ac G G L Y A C(Ace) H M G P I T 1 Nal 119 [ Ac R T R E Y S C Q M G P L T W 120 [ Ac SR A R Y M C H M G P L T W 121 [ Ac G G R A Y M C R L G P V T W 122 [ AcT I A Q Y I C Y M G P E T W 123 [ Ac N G R T Y S C Q L G P V T W 122 [Ac T I A Q Y I C Y M G P E T W 123 [ Ac N G R T Y S C Q L G P V T W 124[ Ac M R T R Y R C Y M G P L T W 125 [ Ac S R T R Y R C E M G P L T W126 [ Ac G S R T Y S C Q L G P V T W 127 [ Ac R P R P Y S C T M G P R TW 128 [ Ac G G T Y S C H F G P L T W 129 [ Ac G G D Y H C R M G P L T W130 [ Ac G G V Y A C R M G P I T W 131 [ Ac G G L Y A C H M G P I Y 1Nal 132 [ Ac G G L Y A C H M G P I T 1 Nal 133 [ Ac G G L Y A C H M G PI T 1 Nal 134 [ Ac G G L Y A C H M G P I t 1 Nal 135 [ Ac G G L Y A C HM G P I T 1 Nal 136 [ Ac G G L y A C H M G P I T 1 Nal 137 [ Ac G G L YA C H M G P I T 1 Nal 138 [ Ac G G L Y A C H M G P I T 1 Nal 139 [ Ac GG L Y A C H M G P I T 1 Nal 140 [ Ac G G G L Y A C H M G P I T 1 Nal 141[ Ac G G L L Y A C H M G P I T 1 Nal 142 [ Ac G G L Y Y A C H M G P I T1 Nal 143 [ Ac G G L Y A A C H M G P I T 1 Nal 144 [ Ac G G L Y A C H HM G P I T 145 [ Ac G G L Y A C H M M G P I T 146 [ Ac N Y T C R F G P LT W 147 [ Ac S W D C R I G P I T W 148 [ Ac N Y M C H F G P I T W 149 [Ac L Y L C R M G P Q T W 150 [ Ac W Y S C L M G P M T W 151 [ Ac E Y F CR M G P I T W 152 [ Ac G G L Y A C H M G G P I T 153 [ Ac G G L Y A C HM G P I I T 154 [ Ac G G L Y A C H M G P I T T 155 [ Ac G G L Y A C H MG P I T 1 Nal 156 [ Ac G G L Y A C H M G P I T 1 Nal 157 [ Ac G G L Y AC H M G P I T 1 Nal 158 [ Ac G G L Y A C H M G P I T 1 Nal 159 [ Ac G GL Y A C H M G P I T 1 Nal 160 [ Ac G G L Y A C H M G P I T 1 Nal SEQ IDNO: Linker 34 V C Q P L R Ser K NH₂ Monomer 35 V C Q P L R Ser

NH₂ ]2 IDA H2 Dimer 36 V C Q P L R Ser

NH₂ ]2 IDA + Boc Boc on IDA 37 V C Q P L R Ser

NH₂ ]2 IDA PEG 38 39 V C Q P L R Ser

NH₂ ]2 IDA Bis sulfone 40 V C Q P L R Ser

NH₂ ]2 IDA Bis sulfone 41 V C Q P L R Ser

NH₂ ]2 IDA Bis sulfoxide, asymmetric 42 V C Q P L R Ser

NH₂ ]2 IDA Bis sulfoxide, asymmetric 43 V C Q P L R Ser

NH₂ ]2 IDA Mono sulfoxide, asymmetric 44 V C Q P L R Ser

NH₂ ]2 IDA Mono sulfoxide, asymmetric 45 V C Q P L R Ser

NH₂ ]2 IDA Mono sulfoxide, asymmetric 46 V C Q P L R Ser NH₂ ]2 IDAN-terminal dimer 47 V C Q P L R Ser NH₂ ]2 IDA N-terminal dimer 48 V C QP L R Ser

NH₂ ]2 IDA Parallel disulfide 49 V C Q P L R Ser

NH₂ ]2 IDA Bis sulfoxide 50 V C Q P L R Ser

NH₂ ]2 IDA Crisscross disulfide 51 V C R R W

NH₂ ]2 IDA + Boc 52 V C S T A L R

NH₂ ]2 IDA + Boc 53 V C R R R R R V

NH₂ ]2 IDA + Boc 54 V C T P S R R R

NH₂ ]2 IDA + Boc 55 V C Q P A R R D

NH₂ ]2 IDA + Boc 56 V C G R R R

NH₂ ]2 IDA + Boc 57 E C A P I R P R K NH₂ Monomer 58 V C E R K NH₂Monomer 59 E C R P R R R E K NH₂ Monomer 60 V C R P L S G R K NH₂Monomer 61 V C A P R R S A L T K NH₂ Monomer 62 V C Q P T R I H K NH₂Monomer 63 E C L P I R P R

NH₂ ]2 IDA + Boc 64 V C E R

NH₂ ]2 IDA + Boc 65 E C R P R R R E

NH₂ ]2 IDA + Boc 66 V C R P L S G R

NH₂ ]2 IDA + Boc 67 V C A P R R S A L T

NH₂ ]2 IDA + Boc 68 V C Q P T R I H

NH₂ ]2 IDA + Boc 69 V C S S R G T Q

NH₂ ]2 IDA + Boc 70 V C A P N R

NH₂ ]2 IDA + Boc 71 V C Q P A R R D

NH₂ ]2 IDA + Boc 72 V C R S S R P R

NH₂ ]2 IDA + Boc 73 V C E G S R L

NH₂ ]2 IDA + Boc 74 V C R P R R S L

NH₂ ]2 IDA + Boc 75 V C P D S Y E F

NH₂ ]2 IDA + Boc 76 V E C D D I S K NH₂ Monomer 77 E C M R A A G V T

NH₂ ]2 IDA + Boc 78 V C G R D R H L

NH₂ ]2 IDA + Boc 79 V C S S R G T Q K NH₂ Monomer 80 V C A P N R K NH₂Monomer 81 V C Q P A R R D K NH₂ Monomer 82 V C R S S R P R K NH₂Monomer 83 V C E G S R L K NH₂ Monomer 84 V C R P R R S L K NH₂ Monomer85 V C P D S Y E F K NH₂ Monomer 86 V C G G V R A G K NH₂ Monomer 87 E CM R A A G V T K NH₂ Monomer 88 C G R D R H L K NH₂ Monomer 89 V C Q P LR Ser

NH₂ ]2 IDA Monosulfoxide 90 V C Q P L R Ser

NH₂ ]2 IDA Monosulfoxide 91 V C Q P L R Ser

NH₂ ]2 IDA D Leu 92 V C Q P L R Ser

NH₂ ]2 IDA D Met 93 V c Q P L R Ser

NH₂ ]2 IDA 2 D cys 94 V C Q P L R Ser

NH₂ ]2 IDA D cys 95 V c Q P L R Ser

NH₂ ]2 IDA D cys 96 V C Q p L R Ser

NH₂ ]2 IDA D Pro 97 V C E R Y

NH₂ ]2 IDA + Boc 98 V C R P Q

NH₂ ]2 IDA + Boc 99 V C R P L

NH₂ ]2 IDA + Boc 100 V C R P G

NH₂ ]2 IDA + Boc 101 E C Q P R

NH₂ ]2 IDA + Boc 102 V C R P S

NH₂ ]2 IDA + Boc 103 L C T A Q

NH₂ ]2 IDA 104 V C S P T

NH₂ ]2 IDA 105 D C G Y

NH₂ ]2 IDA 106 V C T P D

NH₂ ]2 IDA 107 V C Q P L R Ser

NH₂ ]2 IDA D Leu 108 V C Q P L R Ser NH₂ ]2 IDA N-terminal dimer 109 V CQ P L R Ser NH₂ ]2 IDA Parallel disulfide 110 V C Q P L R Ser

NH₂ ]2 IDA D Pro 111 V C Q P L R Ser

NH₂ ]2 IDA D His 112 V C Q P L R Ser

NH₂ ]2 IDA D Ala 113 V Cxx Q P L R Ser

NH₂ ]2 IDA 1/2 Crisscross disulfide 114 V C Q P L R Ser

NH₂ ]2 IDA 1/2 Parallel disulfide 115 V C Q P L R Ser NH₂ ]2 IDAN-terminal dimer 116 V C Q P L R Ser

NH₂ ]2 IDA Crisscross disulfide 117 V CSH Q P L R Ser

NH₂ ]2 IDA Reduced disulfide 118 V C (Ace) Q P L R Ser

NH₂ ]2 IDA Capped Cysteine, no SS 119 T C V P R S

NH₂ ]2 IDA + Boc 120 V C R P E V

NH₂ ]2 IDA + Boc 121 V C S P R I R I

NH₂ ]2 IDA + Boc 122 E C R P S P R A

NH₂ ]2 IDA + Boc 123 V C S R G V R R

NH₂ ]2 IDA + Boc 122 E C R P S P R A

NH₂ ]2 IDA + Boc 123 V C S R G V R R

NH₂ ]2 IDA + Boc 124 V C E G S R L

NH₂ ]2 IDA + Boc 125 V C E R W

NH₂ ]2 IDA + Boc 126 V C G R R R

NH₂ ]2 IDA + Boc 127 V C G G V R A G

NH₂ ]2 IDA + Boc 128 V C R P Q G G

NH₂ ]2 IDA + Boc 129 V C R P L G G

NH₂ ]2 IDA + Boc 130 V C S P L G G

NH₂ ]2 IDA 131 V C E P L R Ser

NH₂ ]2 IDA Deamidated 132 V C E P L R Ser

OH ]2 IDA Bis-Deamidated 133 V C Q P L R Ser k NH2 ]2 IDA D Lys 134 V CQ P L R Ser

NH₂ ]2 IDA D Thr 135 V C q P L R Ser

NH₂ ]2 IDA D Gln 136 V C Q P L R Ser

NH₂ ]2 IDA D Tyr 137 V C Q P L R Ser

NH₂ ]2 IDA D Ile 138 v C Q P L R Ser

NH₂ ]2 IDA D Val 139 V C Q P L R Ser

NH₂ ]2 IDA D 1 Nal 140 V C Q P L R Ser

NH₂ ]2 IDA Gly Insertion 141 V C Q P L R Ser

NH₂ ]2 IDA Leu Insertion 142 V C Q P L R Ser

NH₂ ]2 IDA Tyr Insertion 143 V C Q P L R Ser

NH₂ ]2 IDA Ala Insertion 144 1 Nal V C Q P L R Ser

NH₂ ]2 IDA His Insertion 145 1 Nal V C Q P L R Ser

NH₂ ]2 IDA Met Insertion 146 E C T P Q

NH₂ ]2 IDA- Boc 147 V C R W S

NH₂ ]2 IDA- Boc 148 V C R P G

NH₂ ]2 IDA- Boc 149 M C Q P G

NH₂ ]2 IDA- Boc 150 V C R A H

NH₂ ]2 IDA- Boc 151 V C Q R S

NH₂ ]2 IDA- Boc 152 1 Nal V C Q P L R Ser

NH₂ ]2 IDA Gly Insertion 153 1 Nal V C Q P L R Ser

NH₂ ]2 IDA Ile Insertion 154 1 Nal V C Q P L R Ser

NH₂ ]2 IDA Thr Insertion 155 V V C Q P L R Ser

NH₂ ]2 IDA Val Insertion 156 V C Q Q P L R Ser

NH₂ ]2 IDA Gln Insertion 157 V C Q P P L R Ser

NH₂ ]2 IDA Pro Insertion 158 V C Q P L L R Ser

NH₂ ]2 IDA Leu Insertion 159 V C Q P L R R Ser

NH₂ ]2 IDA Arg Insertion 160 V C Q P L R Ser Ser

NH₂ ]2 IDA Sar Insertion Branching amino acid to linker shown shaded IDAis imino diacetic acid (BetaAla conjugate)

The peptide sequences of the present invention can be present alone orin conjunction with N-terminal and/or C-terminal extensions of thepeptide chain. Such extensions may be naturally encoded peptidesequences optionally with or substantially without non-naturallyoccurring sequences; the extensions may include any additions,deletions, point mutations, or other sequence modifications orcombinations as desired by those skilled in the art. For example and notlimitation, naturally-occurring sequences may be full-length or partiallength and may include amino acid substitutions to provide a site forattachment of carbohydrate, PEG, other polymer, or the like via sidechain conjugation. In a variation, the amino acid substitution resultsin humanization of a sequence to make in compatible with the humanimmune system. Fusion proteins of all types are provided, includingimmunoglobulin sequences adjacent to or in near proximity to the EPO-Ractivating sequences of the present invention with or without anon-immunoglobulin spacer sequence. One type of embodiment is animmunoglobulin chain having the EPO-R activating sequence in place ofthe variable (V) region of the heavy and/or light chain.

Preparation of the Peptide Compounds of the Invention: Peptide Synthesis

The peptides of the invention may be prepared by classical methods knownin the art. These standard methods include exclusive solid phasesynthesis, partial solid phase synthesis methods, fragment condensation,classical solution synthesis, and recombinant DNA technology [See, e.g.,Merrifield J. Am. Chem. Soc. 1963 85:2149].

In one embodiment, the peptide monomers of a peptide dimer aresynthesized individually and dimerized subsequent to synthesis. Inpreferred embodiments the peptide monomers of a dimer have the sameamino acid sequence.

In particularly preferred embodiments, the peptide monomers of a dimerare linked via their C-termini by a linker L_(K) moiety having twofunctional groups capable of serving as initiation sites for peptidesynthesis and a third functional group (e.g., a carboxyl group or anamino group) that enables binding to another molecular moiety (e.g., asmay be present on the surface of a solid support). In this case, the twopeptide monomers may be synthesized directly onto two reactive nitrogengroups of the linker L_(K) moiety in a variation of the solid phasesynthesis technique. Such synthesis may be sequential or simultaneous.

Where sequential synthesis of the peptide chains of a dimer onto alinker is to be performed, two amine functional groups on the linkermolecule are protected with two different orthogonally removable amineprotecting groups. In preferred embodiments, the protected diamine is aprotected lysine. The protected linker is coupled to a solid support viathe linker's third functional group. The first amine protecting group isremoved, and the first peptide of the dimer is synthesized on the firstdeprotected amine moiety. Then the second amine protecting group isremoved, and the second peptide of the dimer is synthesized on thesecond deprotected amine moiety. For example, the first amino moiety ofthe linker may be protected with Alloc, and the second with Fmoc. Inthis case, the Fmoc group (but not the Alloc group) may be removed bytreatment with a mild base [e.g., 20% piperidine in dimethyl formamide(DMF)], and the first peptide chain synthesized. Thereafter the Allocgroup may be removed with a suitable reagent [e.g., Pd(PPh₃)/4-methylmorpholine and chloroform], and the second peptide chain synthesized.This technique may be used to generate dimers wherein the sequences ofthe two peptide chains are identical or different. Note that wheredifferent thiol-protecting groups for cysteine are to be used to controldisulfide bond formation (as discussed below) this technique must beused even where the final amino acid sequences of the peptide chains ofa dimer are identical.

Where simultaneous synthesis of the peptide chains of a dimer onto alinker is to be performed, two amine functional groups of the linkermolecule are protected with the same removable amine protecting group.In preferred embodiments, the protected diamine is a protected lysine.The protected linker is coupled to a solid support via the linker'sthird functional group. In this case the two protected functional groupsof the linker molecule are simultaneously deprotected, and the twopeptide chains simultaneously synthesized on the deprotected amines.Note that using this technique, the sequences of the peptide chains ofthe dimer will be identical, and the thiol-protecting groups for thecysteine residues are all the same.

A preferred method for peptide synthesis is solid phase synthesis. Solidphase peptide synthesis procedures are well-known in the art [see, e.g.,Stewart Solid Phase Peptide Syntheses (Freeman and Co.: San Francisco)1969; 2002/2003 General Catalog from Novabiochem Corp, San Diego, USA;Goodman Synthesis of Peptides and Peptidomimetics (Houben-Weyl,Stuttgart) 2002]. In solid phase synthesis, synthesis is typicallycommenced from the C-terminal end of the peptide using an α-aminoprotected resin. A suitable starting material can be prepared, forinstance, by attaching the required α-amino acid to a chloromethylatedresin, a hydroxymethyl resin, a polystyrene resin, a benzhydrylamineresin, or the like. One such chloromethylated resin is sold under thetrade name BIO-BEADS SX-1 by Bio Rad Laboratories (Richmond, Calif.).The preparation of the hydroxymethyl resin has been described[Bodonszky, et al. (1966) Chem. Ind. London 38:1597]. Thebenzhydrylamine (BHA) resin has been described [Pietta and Marshall(1970) Chem. Commun. 650], and the hydrochloride form is commerciallyavailable from Beckman Instruments, Inc. (Palo Alto, Calif.). Forexample, an α-amino protected amino acid may be coupled to achloromethylated resin with the aid of a cesium bicarbonate catalyst,according to the method described by Gisin (1973) Helv. Chim. Acta56:1467.

After initial coupling, the α-amino protecting group is removed, forexample, using trifluoroacetic acid (TFA) or hydrochloric acid (HCl)solutions in organic solvents at room temperature. Thereafter, α-aminoprotected amino acids are successively coupled to a growingsupport-bound peptide chain. The α-amino protecting groups are thoseknown to be useful in the art of stepwise synthesis of peptides,including: acyl-type protecting groups (e.g., formyl, trifluoroacetyl,acetyl), aromatic urethane-type protecting groups [e.g.,benzyloxycarboyl (Cbz) and substituted Cbz], aliphatic urethaneprotecting groups [e.g., t-butyloxycarbonyl (Boc), isopropyloxycarbonyl,cyclohexyloxycarbonyl], and alkyl type protecting groups (e.g., benzyl,triphenylmethyl), fluorenylmethyl oxycarbonyl (Fmoc), allyloxycarbonyl(Alloc), and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde).

The side chain protecting groups (typically ethers, esters, trityl, PMC,and the like) remain intact during coupling and is not split off duringthe deprotection of the amino-terminus protecting group or duringcoupling. The side chain protecting group must be removable upon thecompletion of the synthesis of the final peptide and under reactionconditions that will not alter the target peptide. The side chainprotecting groups for Tyr include tetrahydropyranyl, tert-butyl, trityl,benzyl, Cbz, Z-Br-Cbz, and 2,5-dichlorobenzyl. The side chain protectinggroups for Asp include benzyl, 2,6-dichlorobenzyl, methyl, ethyl, andcyclohexyl. The side chain protecting groups for Thr and Ser includeacetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl,and Cbz. The side chain protecting groups for Arg include nitro, Tosyl(Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts),2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf),4-mthoxy-2,3,6-trimethyl-benzenesulfonyl (Mtr), or Boc. The side chainprotecting groups for Lys include Cbz, 2-chlorobenzyloxycarbonyl(2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-Br-Cbz), Tos, or Boc.

After removal of the α-amino protecting group, the remaining protectedamino acids are coupled stepwise in the desired order. Each protectedamino acid is generally reacted in about a 3-fold excess using anappropriate carboxyl group activator such as2-(1H-benzotriazol-1-yl)-1,1,3,3 tetramethyluronium hexafluorophosphate(HBTU) or dicyclohexylcarbodimide (DCC) in solution, for example, inmethylene chloride (CH₂Cl₂), N-methylpyrrolidone, dimethyl formamide(DMF), or mixtures thereof.

After the desired amino acid sequence has been completed, the desiredpeptide is decoupled from the resin support by treatment with a reagent,such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF), which notonly cleaves the peptide from the resin, but also cleaves all remainingside chain protecting groups. When a chloromethylated resin is used,hydrogen fluoride treatment results in the formation of the free peptideacids. When the benzhydrylamine resin is used, hydrogen fluoridetreatment results directly in the free peptide amide. Alternatively,when the chloromethylated resin is employed, the side chain protectedpeptide can be decoupled by treatment of the peptide resin with ammoniato give the desired side chain protected amide or with an alkylamine togive a side chain protected alkylamide or dialkylamide. Side chainprotection is then removed in the usual fashion by treatment withhydrogen fluoride to give the free amides, alkylamides, ordialkylamides. In preparing the esters of the invention, the resins usedto prepare the peptide acids are employed, and the side chain protectedpeptide is cleaved with base and the appropriate alcohol (e.g.,methanol). Side chain protecting groups are then removed in the usualfashion by treatment with hydrogen fluoride to obtain the desired ester.

These procedures can also be used to synthesize peptides in which aminoacids other than the 20 naturally occurring, genetically encoded aminoacids are substituted at one, two, or more positions of any of thecompounds of the invention. Synthetic amino acids that can besubstituted into the peptides of the present invention include, but arenot limited to, N-methyl, L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl,δ amino acids such as L-δ-hydroxylysyl and D-δ-methylalanyl,L-α-methylalanyl, β amino acids, and isoquinolyl. D-amino acids andnon-naturally occurring synthetic amino acids can also be incorporatedinto the peptides of the present invention.

Peptide Modifications

One can also modify the amino and/or carboxy termini of the peptidecompounds of the invention to produce other compounds of the invention.Amino terminus modifications include methylation (e.g., —NHCH₃ or—N(CH₃)₂), acetylation (e.g., with acetic acid or a halogenatedderivative thereof such as α-chloroacetic acid, α-bromoacetic acid, orα-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blockingthe amino terminus with any blocking group containing a carboxylatefunctionality defined by RCOO— or sulfonyl functionality defined byR—SO₂—, where R is selected from alkyl, aryl, heteroaryl, alkyl aryl,and the like, and similar groups. One can also incorporate a desaminoacid at the N-terminus (so that there is no N-terminal amino group) todecrease susceptibility to proteases or to restrict the conformation ofthe peptide compound. In preferred embodiments, the N-terminus isacetylated. In particularly preferred embodiments an N-terminal glycineis acetylated to yield N-acetylglycine (AcG).

Carboxy terminus modifications include replacing the free acid with acarboxamide group or forming a cyclic lactam at the carboxy terminus tointroduce structural constraints. One can also cyclize the peptides ofthe invention, or incorporate a desamino or descarboxy residue at thetermini of the peptide, so that there is no terminal amino or carboxylgroup, to decrease susceptibility to proteases or to restrict theconformation of the peptide. C-terminal functional groups of thecompounds of the present invention include amide, amide lower alkyl,amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lowerester derivatives thereof, and the pharmaceutically acceptable saltsthereof.

One can replace the naturally occurring side chains of the 20genetically encoded amino acids (or the stereoisomeric D amino acids)with other side chains, for instance with groups such as alkyl, loweralkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl,amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lowerester derivatives thereof, and with 4-, 5-, 6-, to 7-memberedheterocyclic. In particular, proline analogues in which the ring size ofthe proline residue is changed from 5 members to 4, 6, or 7 members canbe employed. Cyclic groups can be saturated or unsaturated, and ifunsaturated, can be aromatic or non-aromatic. Heterocyclic groupspreferably contain one or more nitrogen, oxygen, and/or sulfurheteroatoms. Examples of such groups include the furazanyl, furyl,imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl,morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g.,1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g.,thiomorpholino), and triazolyl. These heterocyclic groups can besubstituted or unsubstituted. Where a group is substituted, thesubstituent can be alkyl, alkoxy, halogen, oxygen, or substituted orunsubstituted phenyl.

One can also readily modify peptides by phosphorylation, and othermethods [e.g., as described in Hruby, et al. (1990) Biochem J.268:249-262].

The peptide compounds of the invention also serve as structural modelsfor non-peptidic compounds with similar biological activity. Those ofskill in the art recognize that a variety of techniques are availablefor constructing compounds with the same or similar desired biologicalactivity as the lead peptide compound, but with more favorable activitythan the lead with respect to solubility, stability, and susceptibilityto hydrolysis and proteolysis [See, Morgan and Gainor (1989) Ann. Rep.Med. Chem. 24:243-252]. These techniques include replacing the peptidebackbone with a backbone composed of phosphonates, amidates, carbamates,sulfonamides, secondary amines, and N-methylamino acids.

Formation of Disulfide Bonds

The compounds of the present invention may contain one or moreintramolecular disulfide bonds. In one embodiment, embodiment, a peptidemonomer or dimer comprises at least one intramolecular disulfide bond.In preferred embodiments, a peptide dimer comprises two intramoleculardisulfide bonds.

Such disulfide bonds may be formed by oxidation of the cysteine residuesof the peptide core sequence. In one embodiment the control of cysteinebond formation is exercised by choosing an oxidizing agent of the typeand concentration effective to optimize formation of the desired isomer.For example, oxidation of a peptide dimer to form two intramoleculardisulfide bonds (one on each peptide chain) is preferentially achieved(over formation of intermolecular disulfide bonds) when the oxidizingagent is DMSO.

In preferred embodiments, the formation of cysteine bonds is controlledby the selective use of thiol-protecting groups during peptidesynthesis. For example, where a dimer with two intramolecular disulfidebonds is desired, the first monomer peptide chain is synthesized withthe two cysteine residues of the core sequence protected with a firstthiol protecting group [e.g., trityl(Trt), allyloxycarbonyl (Alloc), and1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) or the like],then the second monomer peptide is synthesized the two cysteine residuesof the core sequence protected with a second thiol protecting groupdifferent from the first thiol protecting group [e.g., acetamidomethyl(Acm), t-butyl (tBu), or the like]. Thereafter, the first thiolprotecting groups are removed effecting bisulfide cyclization of thefirst monomer, and then the second thiol protecting groups are removedeffecting bisulfide cyclization of the second monomer.

Other embodiments of this invention provide for analogues of thesedisulfide derivatives in which one of the sulfurs has been replaced by aCH₂ group or other isotere for sulfur. These analogues can be preparedfrom the compounds of the present invention, wherein each core sequencecontains at least one C or homocysteine residue and an α-amino-γ-butyricacid in place of the second C residue, via an intramolecular orintermolecular displacement, using methods known in the art [See, e.g.,Barker, et al. (1992) J. Med. Chem. 35:2040-2048 and Or, et al. (1991)J. Org. Chem. 56:3146-3149]. One of skill in the art will readilyappreciate that this displacement can also occur using other homologs ofα-amino-γ-butyric acid and homocysteine.

In addition to the foregoing cyclization strategies, other non-disulfidepeptide cyclization strategies can be employed. Such alternativecyclization strategies include, for example, amide-cyclizationstrategies as well as those involving the formation of thio-ether bonds.Thus, the compounds of the present invention can exist in a cyclizedform with either an intramolecular amide bond or an intramolecularthio-ether bond. For example, a peptide may be synthesized wherein onecysteine of the core sequence is replaced with lysine and the secondcysteine is replaced with glutamic acid. Thereafter a cyclic monomer maybe formed through an amide bond between the side chains of these tworesidues. Alternatively, a peptide may be synthesized wherein onecysteine of the core sequence is replaced with lysine. A cyclic monomermay then be formed through a thio-ether linkage between the side chainsof the lysine residue and the second cysteine residue of the coresequence. As such, in addition to disulfide cyclization strategies,amide-cyclization strategies and thio-ether cyclization strategies canboth be readily used to cyclize the compounds of the present invention.Alternatively, the amino-terminus of the peptide can be capped with anα-substituted acetic acid, wherein the α-substituent is a leaving group,such as an α-haloacetic acid, for example, α.-chloroacetic acid,α-bromoacetic acid, or α-iodoacetic acid.

Addition of Linkers

In embodiments where a peptide dimer is dimerized by a linker L_(K)moiety, said linker may be incorporated into the peptide during peptidesynthesis. For example, where a linker L_(K) moiety contains twofunctional groups capable of serving as initiation sites for peptidesynthesis and a third functional group (e.g., a carboxyl group or anamino group) that enables binding to another molecular moiety, thelinker may be conjugated to a solid support. Thereafter, two peptidemonomers may be synthesized directly onto the two reactive nitrogengroups of the linker L_(K) moiety in a variation of the solid phasesynthesis technique.

In alternate embodiments where a peptide dimer is dimerized by a linkerL_(K) moiety, said linker may be conjugated to the two peptide monomersof a peptide dimer after peptide synthesis. Such conjugation may beachieved by methods well established in the art. In one embodiment, thelinker contains at least two functional groups suitable for attachmentto the target functional groups of the synthesized peptide monomers. Forexample, a linker with two free amine groups may be reacted with theC-terminal carboxyl groups of each of two peptide monomers. In anotherexample, linkers containing two carboxyl groups, either preactivated orin the presence of a suitable coupling reagent, may be reacted with theN-terminal or side chain amine groups, or C-terminal lysine amides, ofeach of two peptide monomers.

Addition of Spacers

In embodiments where the peptide compounds contain a spacer moiety, saidspacer may be incorporated into the peptide during peptide synthesis.For example, where a spacer contains a free amino group and a secondfunctional group (e.g., a carboxyl group or an amino group) that enablesbinding to another molecular moiety, the spacer may be conjugated to thesolid support. Thereafter, the peptide may be synthesized directly ontothe spacer's free amino group by standard solid phase techniques.

In a preferred embodiment, a spacer containing two functional groups isfirst coupled to the solid support via a first functional group. Next alinker L_(K) moiety having two functional groups capable of serving asinitiation sites for peptide synthesis and a third functional group(e.g., a carboxyl group or an amino group) that enables binding toanother molecular moiety is conjugated to the spacer via the spacer'ssecond functional group and the linker's third functional group.Thereafter, two peptide monomers may be synthesized directly onto thetwo reactive nitrogen groups of the linker L_(K) moiety in a variationof the solid phase synthesis technique. For example, a solid supportcoupled spacer with a free amine group may be reacted with a lysinelinker via the linker's free carboxyl group.

In alternate embodiments where the peptide compounds contain a spacermoiety, said spacer may be conjugated to the peptide after peptidesynthesis. Such conjugation may be achieved by methods well establishedin the art. In one embodiment, the linker contains at least onefunctional group suitable for attachment to the target functional groupof the synthesized peptide. For example, a spacer with a free aminegroup may be reacted with a peptide's C-terminal carboxyl group. Inanother example, a linker with a free carboxyl group may be reacted withthe free amine group of a peptide's N-terminus or of a lysine residue.In yet another example, a spacer containing a free sulfhydryl group maybe conjugated to a cysteine residue of a peptide by oxidation to form adisulfide bond.

Attachment of Water Soluble Polymers

In recent years, water-soluble polymers, such as polyethylene glycol(PEG), have been used for the covalent modification of peptides oftherapeutic and diagnostic importance. Attachment of such polymers isthought to enhance biological activity, prolong blood circulation time,reduce immunogenicity, increase aqueous solubility, and enhanceresistance to protease digestion. For example, covalent attachment ofPEG to therapeutic polypeptides such as interleukins [Knauf, et al.(1988) J. Biol. Chem. 263; 15064; Tsutsumi, et al. (1995) J. ControlledRelease 33:447), interferons (Kita, et al. (1990) Drug Des. Delivery6:157), catalase (Abuchowski, et al. (1977) J. Biol. Chem. 252:582),superoxide dismutase (Beauchamp, et al. (1983) Anal. Biochem. 131:25),and adenosine deaminase (Chen, et al. (1981) Biochim. Biophy. Acta660:293), has been reported to extend their half life in vivo, and/orreduce their immunogenicity and antigenicity.

The peptide compounds of the invention may further comprise one or morewater soluble polymer moieties. Preferably, these polymers arecovalently attached to the peptide compounds. The water soluble polymermay be, for example, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol,propropylene glycol homopolymers, polypropylene oxide/ethylene oxidecopolymers, and polyoxyethylated polyols. A preferred water solublepolymer is PEG.

Peptides, peptide dimers and other peptide-based molecules of theinvention can be attached to water-soluble polymers (e.g., PEG) usingany of a variety of chemistries to link the water-soluble polymer(s) tothe receptor-binding portion of the molecule (e.g., peptide+spacer). Atypical embodiment employs a single attachment junction for covalentattachment of the water soluble polymer(s) to the receptor-bindingportion, however in alternative embodiments multiple attachmentjunctions may be used, including further variations wherein differentspecies of water-soluble polymer are attached to the receptor-bindingportion at distinct attachment junctions, which may include covalentattachment junction(s) to the spacer and/or to one or both peptidechains. In some embodiments, the dimer or higher order multimer willcomprise distinct species of peptide chain (i.e., a heterodimer or otherheteromultimer). By way of example and not limitation, a dimer maycomprise a first peptide chain having a PEG attachment junction and thesecond peptide chain may either lack a PEG attachment junction orutilize a different linkage chemistry than the first peptide chain andin some variations the spacer may contain or lack a PEG attachmentjunction and said spacer, if PEGylated, may utilize a linkage chemistrydifferent than that of the first and/or second peptide chains. Analternative embodiment employs a PEG attached to the spacer portion ofthe receptor-binding portion and a different water-soluble polymer(e.g., a carbohydrate) conjugated to a side chain of one of the aminoacids of the peptide portion of the molecule.

A wide variety of polyethylene glycol (PEG) species may be used forPEGylation of the receptor-binding portion (peptides+spacer).Substantially any suitable reactive PEG reagent can be used. Inpreferred embodiments, the reactive PEG reagent will result in formationof a carbamate or amide bond upon conjugation to the receptor-bindingportion. Suitable reactive PEG species include, but are not limited to,those which are available for sale in the Drug Delivery Systems catalog(2003) of NOF Corporation (Yebisu Garden Place Tower, 20-3 Ebisu4-chome, Shibuya-ku, Tokyo 150-6019) and the Molecular Engineeringcatalog (2003) of Nektar Therapeutics (490 Discovery Drive, Huntsville,Ala. 35806). For example and not limitation, the following PEG reagentsare often preferred in various embodiments: mPEG2-NHS, mPEG2-ALD,multi-Arm PEG, mPEG(MAL)₂, mPEG2(MAL), mPEG-NH2, mPEG-SPA, mPEG-SBA,mPEG-thioesters, mPEG-Double Esters, mPEG-BTC, mPEG-ButyrALD, mPEG-ACET,heterofunctional PEGs (NH2-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS,NHS-PEG-VS, NHS-PEG-MAL), PEG acrylates (ACRL-PEG-NHS),PEG-phospholipids (e.g., mPEG-DSPE), multiarmed PEGs of the SUNBRITEseries including the GL series of glycerine-based PEGs activated by achemistry chosen by those skilled in the art, any of the SUNBRITEactivated PEGs (including but not limited to carboxyl-PEGs, p-NP-PEGs,Tresyl-PEGs, aldehyde PEGs, acetal-PEGs, amino-PEGs, thiol-PEGs,maleimido-PEGs, hydroxyl-PEG-amine, amino-PEG-COOH,hydroxyl-PEG-aldehyde, carboxylic anhydride type-PEG, functionalizedPEG-phospholipid, and other similar and/or suitable reactive PEGs asselected by those skilled in the art for their particular applicationand usage.

The polymer may be of any molecular weight, and may be branched orunbranched. A preferred PEG for use in the present invention compriseslinear, unbranched PEG having a molecular weight of from about 20kilodaltons (kD) to about 40 kD (the term “about” indicating that inpreparations of PEG, some molecules will weigh more, some less, than thestated molecular weight). Most preferably, the PEG has a molecularweight of from about 30 kD to about 40 kD. Other sizes may be used,depending on the desired therapeutic profile (e.g., duration ofsustained release desired; effects, if any, on biological activity; easein handling; degree or lack of antigenicity; and other known effects ofPEG on a therapeutic peptide).

The number of polymer molecules attached may vary; for example, one,two, three, or more water soluble polymers may be attached to an EPO-Ragonist peptide of the invention. The multiple attached polymers may bethe same or different chemical moieties (e.g., PEGs of differentmolecular weight). In some cases, the degree of polymer attachment (thenumber of polymer moieties attached to a peptide and/or the total numberof peptides to which a polymer is attached) may be influenced by theproportion of polymer molecules versus peptide molecules in anattachment reaction, as well as by the total concentration of each inthe reaction mixture. In general, the optimum polymer versus peptideratio (in terms of reaction efficiency to provide for no excessunreacted peptides and/or polymer moieties) will be determined byfactors such as the desired degree of polymer attachment (e.g., mono,di-, tri-, etc.), the molecular weight of the polymer selected, whetherthe polymer is branched or unbranched, and the reaction conditions for aparticular attachment method.

In preferred embodiments, the covalently attached water soluble polymeris PEG. For illustrative purposes, examples of methods for covalentattachment of PEG (PEGylation) are described below. These illustrativedescriptions are not intended to be limiting. One of ordinary skill inthe art will appreciate that a variety of methods for covalentattachment of a broad range of water soluble polymers is wellestablished in the art. As such, peptide compounds to which any of anumber of water soluble polymers known in the art have been attached byany of a number of attachment methods known in the art are encompassedby the present invention.

In one embodiment, PEG may serve as a linker that dimerizes two peptidemonomers. In one embodiment, PEG is attached to at least one terminus(N-terminus or C-terminus) of a peptide monomer or dimer. In anotherembodiment, PEG is attached to a spacer moiety of a peptide monomer ordimer. In a preferred embodiment PEG is attached to the linker moiety ofa peptide dimer. In a highly preferred embodiment, PEG is attached to aspacer moiety, where said spacer moiety is attached to the linker L_(K)moiety that connects the monomers of a peptide dimer. Most preferably,PEG is attached to a spacer moiety, where said spacer moiety is attachedto a peptide dimer via the carbonyl carbon of a lysine linker, or theamide nitrogen of a lysine amide linker.

There are a number of PEG attachment methods available to those skilledin the art [see, e.g., Goodson, et al. (1990) Bio/Technology 8:343(PEGylation of interleukin-2 at its glycosylation site aftersite-directed mutagenesis); EP 0 401 384 (coupling PEG to G-CSF); Malik,et al., (1992) Exp. Hematol. 20:1028-1035 (PEGylation of GM-CSF usingtresyl chloride); PCT Pub. No. WO 90/12874 (PEGylation of erythropoietincontaining a recombinantly introduced cysteine residue using acysteine-specific mPEG derivative); U.S. Pat. No. 5,757,078 (PEGylationof EPO peptides); and U.S. Pat. No. 6,077,939 (PEGylation of anN-terminal α-carbon of a peptide)].

For example, PEG may be covalently bound to amino acid residues via areactive group. Reactive groups are those to which an activated PEGmolecule may be bound (e.g., a free amino or carboxyl group). Forexample, N-terminal amino acid residues and lysine (K) residues have afree amino group; and C-terminal amino acid residues have a freecarboxyl group. Sulfhydryl groups (e.g., as found on cysteine residues)may also be used as a reactive group for attaching PEG. In addition,enzyme-assisted methods for introducing activated groups (e.g.,hydrazide, aldehyde, and aromatic-amino groups) specifically at theC-terminus of a polypeptide have been described [Schwarz, et al. (1990)Methods Enzymol. 184:160; Rose, et al. (1991) Bioconjugate Chem. 2:154;Gaertner, et al. (1994) J. Biol. Chem. 269:7224].

For example, PEG molecules may be attached to peptide amino groups usingmethoxylated PEG (“mPEG”) having different reactive moieties. Suchpolymers include mPEG-succinimidyl succinate, mPEG-succinimidylcarbonate, mPEG-imidate, mPEG-4-nitrophenyl carbonate, and mPEG-cyanuricchloride. Similarly, PEG molecules may be attached to peptide carboxylgroups using methoxylated PEG with a free amine group (mPEG-NH₂).

Where attachment of the PEG is non-specific and a peptide containing aspecific PEG attachment is desired, the desired PEGylated compound maybe purified from the mixture of PEGylated compounds. For example, if anN-terminally PEGylated peptide is desired, the N-terminally PEGylatedform may be purified from a population of randomly PEGylated peptides(i.e., separating this moiety from other monoPEGylated moieties).

In preferred embodiments, PEG is attached site-specifically to apeptide. Site-specific PEGylation at the N-terminus, side chain, andC-terminus of a potent analog of growth hormone-releasing factor hasbeen performed through solid-phase synthesis [Felix, et al. (1995) Int.J. Peptide Protein Res. 46:253]. Another site-specific method involvesattaching a peptide to extremities of liposomal surface-grafted PEGchains in a site-specific manner through a reactive aldehyde group atthe N-terminus generated by sodium periodate oxidation of N-terminalthreonine [Zalipsky, et al. (1995) Bioconj. Chem. 6:705]. However, thismethod is limited to polypeptides with N-terminal serine or threonineresidues. Another site-specific method for N-terminal PEGylation of apeptide via a hydrazone, reduced hydrazone, oxime, or reduced oxime bondis described in U.S. Pat. No. 6,077,939 to Wei, et al.

In one method, selective N-terminal PEGylation may be accomplished byreductive alkylation which exploits differential reactivity of differenttypes of primary amino groups (lysine versus the N-terminal) availablefor derivatization in a particular protein. Under the appropriatereaction conditions, a carbonyl group containing PEG is selectiveattached to the N-terminus of a peptide. For example, one mayselectively N-terminally PEGylate the protein by performing the reactionat a pH which exploits the pK_(a) differences between the ε-amino groupsof a lysine residue and the α-amino group of the N-terminal residue ofthe peptide. By such selective attachment, PEGylation takes placepredominantly at the N-terminus of the protein, with no significantmodification of other reactive groups (e.g., lysine side chain aminogroups). Using reductive alkylation, the PEG should have a singlereactive aldehyde for coupling to the protein (e.g., PEGproprionaldehyde may be used).

Site-specific mutagenesis is a further approach which may be used toprepare peptides for site-specific polymer attachment. By this method,the amino acid sequence of a peptide is designed to incorporate anappropriate reactive group at the desired position within the peptide.For example, WO 90/12874 describes the site-directed PEGylation ofproteins modified by the insertion of cysteine residues or thesubstitution of other residues for cysteine residues. This publicationalso describes the preparation of mPEG-erythropoietin (“mPEG-EPO”) byreacting a cysteine-specific mPEG derivative with a recombinantlyintroduced cysteine residue on EPO.

Where PEG is attached to a spacer or linker moiety, similar attachmentmethods may be used. In this case, the linker or spacer contains areactive group and an activated PEG molecule containing the appropriatecomplementary reactive group is used to effect covalent attachment. Inpreferred embodiments the linker or spacer reactive group contains aterminal amino group (i.e., positioned at the terminus of the linker orspacer) which is reacted with a suitably activated PEG molecule to makea stable covalent bond such as an amide or a carbamate. Suitableactivated PEG species include, but are not limited to,mPEG-para-nitrophenylcarbonate (mPEG-NPC), mPEG-succinimidyl carbonate(mPEG-SC), and mPEG-succinimidyl propionate (mPEG-SPA). In otherpreferred embodiments, the linker or spacer reactive group contains acarboxyl group capable of being activated to form a covalent bond withan amine-containing PEG molecule under suitable reaction conditions.Suitable PEG molecules include mPEG-NH₂ and suitable reaction conditionsinclude carbodiimide-mediated amide formation or the like.

EPO-R Agonist Activity Assays: In Vitro Functional Assays

In vitro competitive binding assays quantitate the ability of a testpeptide to compete with EPO for binding to EPO-R. For example (see,e.g., as described in U.S. Pat. No. 5,773,569), the extracellular domainof the human EPO-R (EPO binding protein, EBP) may be recombinantlyproduced in E. coli and the recombinant protein coupled to a solidsupport, such as a microtitre dish or a synthetic bead [e.g., Sulfolinkbeads from Pierce Chemical Co. (Rockford, Ill.)]. Immobilized EBP isthen incubated with labeled recombinant EPO, or with labeled recombinantEPO and a test peptide. Serial dilutions of test peptide are employedfor such experiments. Assay points with no added test peptide definetotal EPO binding to EBP. For reactions containing test peptide, theamount of bound EPO is quantitated and expressed as a percentage of thecontrol (total=100%) binding. These values are plotted versus peptideconcentration. The IC₅₀ value is defined as the concentration of testpeptide which reduces the binding of EPO to EBP by 50% (i.e., 50%inhibition of EPO binding).

A different in vitro competitive binding assay measures the light signalgenerated as a function of the proximity of two beads: an EPO-conjugatedbead and an EPO-R-conjugated bead. Bead proximity is generated by thebinding of EPO to EPO-R. A test peptide that competes with EPO forbinding to EPO-R will prevent this binding, causing a decrease in lightemission. The concentration of test peptide that results in a 50%decrease in light emission is defined as the 1050 value.

The peptides of the present invention compete very efficiently with EPOfor binding to the EPO-R. This enhanced function is represented by theirability to inhibit the binding of EPO at substantially lowerconcentrations of peptide (i.e., they have very low 1050 values).

The biological activity and potency of monomeric and dimeric peptideEPO-R agonists of the invention, which bind specifically to theEPO-receptor, may be measured using in vitro cell-based functionalassays.

One assay is based upon a murine pre-B-cell line expressing human EPO-Rand further transfected with a fos promoter-driven luciferase reportergene construct. Upon exposure to EPO or another EPO-R agonist, suchcells respond by synthesizing luciferase. Luciferase causes the emissionof light upon addition of its substrate luciferin. Thus, the level ofEPO-R activation in such cells may be quantitated via measurement ofluciferase activity. The activity of a test peptide is measured byadding serial dilutions of the test peptide to the cells, which are thenincubated for 4 hours. After incubation, luciferin substrate is added tothe cells, and light emission is measured. The concentration of testpeptide that results in a half-maximal emission of light is recorded asthe EC50.

The peptides of the present invention show dramatically enhanced abilityto promote EPO-R signaling-dependent luciferase expression in thisassay. This enhanced function is represented by their ability to yieldhalf of the maximal luciferase activity at substantially lowerconcentrations of peptide (i.e., they have very low EC50 values). Thisassay is a preferred method for estimating the potency and activity ofan EPO-R agonist peptide of the invention.

Another assay may be performed using FDC-P1/ER cells [Dexter, et al.(1980) J. Exp. Med. 152:1036-1047], a well characterized nontransformedmurine bone marrow derived cell line into which EPO-R has been stablytransfected. These cells exhibit EPO-dependent proliferation.

In one such assay, the cells are grown to half stationary density in thepresence of the necessary growth factors (see, e.g., as described inU.S. Pat. No. 5,773,569). The cells are then washed in PBS and starvedfor 16-24 hours in whole media without the growth factors. Afterdetermining the viability of the cells (e.g., by trypan blue staining),stock solutions (in whole media without the growth factors) are made togive about 10⁵ cells per 504. Serial dilutions of the peptide EPO-Ragonist compounds (typically the free, solution phase peptide as opposedto a phage-bound or other bound or immobilized peptide) to be tested aremade in 96-well tissue culture plates for a final volume of 50 μL perwell. Cells (50 μL) are added to each well and the cells are incubated24-48 hours, at which point the negative controls should die or bequiescent. Cell proliferation is then measured by techniques known inthe art, such as an MTT assay which measures H³-thymidine incorporationas an indication of cell proliferation [see, Mosmann (1983) J. Immunol.Methods 65:55-63]. Peptides are evaluated on both the EPO-R-expressingcell line and a parental non-expressing cell line. The concentration oftest peptide necessary to yield one half of the maximal cellproliferation is recorded as the EC50.

The peptides of the present invention show dramatically enhanced abilityto promote EPO-dependent cell growth in this assay. This enhancedfunction is represented by their ability to yield half of the maximalcell proliferation stimulation activity at substantially lowerconcentrations of peptide (i.e., they have very low EC50 values). Thisassay is a preferred method for estimating the potency and activity ofan EPO-R agonist peptide of the invention.

In another assay, the cells are grown to stationary phase inEPO-supplemented medium, collected, and then cultured for an additional18 hr in medium without EPO. The cells are divided into three groups ofequal cell density: one group with no added factor (negative control), agroup with EPO (positive control), and an experimental group with thetest peptide. The cultured cells are then collected at various timepoints, fixed, and stained with a DNA-binding fluorescent dye (e.g.,propidium iodide or Hoechst dye, both available from Sigma).Fluorescence is then measured, for example, using a FACS Scan Flowcytometer. The percentage of cells in each phase of the cell cycle maythen be determined, for example, using the SOBR model of CellFITsoftware (Becton Dickinson). Cells treated with EPO or an active peptidewill show a greater proportion of cells in S phase (as determined byincreased fluorescence as an indicator of increased DNA content)relative to the negative control group.

Similar assays may be performed using FDCP-1 [see, e.g., Dexter et al.(1980) J. Exp. Med. 152:1036-1047] or TF-1 [Kitamura, et al. (1989)Blood 73:375-380] cell lines. FDCP-1 is a growth factor dependent murinemulti-potential primitive hematopoietic progenitor cell line that canproliferate, but not differentiate, when supplemented withWEHI-3-conditioned media (a medium that contains IL-3, ATCC numberTIB-68). For such experiments, the FDCP-1 cell line is transfected withthe human or murine EPO-R to produce FDCP-1-hEPO-R or FDCP-1-mEPO-R celllines, respectively, that can proliferate, but not differentiate, in thepresence of EPO. TF-1, an EPO-dependent cell line, may also be used tomeasure the effects of peptide EPO-R agonists on cellular proliferation.

In yet another assay, the procedure set forth in Krystal (1983) Exp.Hematol 11:649-660 for a microassay based on H³-thymidine incorporationinto spleen cells may be employed to ascertain the ability of thecompounds of the present invention to serve as EPO agonists. In brief,B6C3F₁ mice are injected daily for two days with phenylhydrazine (60mg/kg). On the third day, spleen cells are removed and their ability toproliferate over a 24 hour period ascertained using an MIT assay.

The binding of EPO to EPO-R in an erythropoietin-responsive cell lineinduces tyrosine phosphorylation of both the receptor and numerousintracellular proteins, including Shc, vav and JAK2 kinase. Therefore,another in vitro assay measures the ability of peptides of the inventionto induce tyrosine phosphorylation of EPO-R and downstream intracellularsignal transducer proteins. Active peptides, as identified by bindingand proliferation assays described above, elicit a phosphorylationpattern nearly identical to that of EPO in erythropoietin-responsivecells. For this assay, FDC-P1/ER cells [Dexter, et al. (1980) J Exp Med152:1036-47] are maintained in EPO-supplemented medium and grown tostationary phase. These cells are then cultured in medium without EPOfor 24 hr. A defined number of such cells is then incubated with a testpeptide for approximately 10 min at 37° C. A control sample of cellswith EPO is also run with each assay. The treated cells are thencollected by centrifugation, resuspended in SDS lysis buffer, andsubjected to SDS polyacrylamide gel electrophoresis. The electrophoresedproteins in the gel are transferred to nitrocellulose, and thephosphotyrosine containing proteins on the blot visualized by standardimmunological techniques. For example, the blot may be probed with ananti-phosphotyrosine antibody (e.g., mouse anti-phosphotyrosine IgG fromUpstate Biotechnology, Inc.), washed, and then probed with a secondaryantibody [e.g., peroxidase labeled goat anti-mouse IgG from Kirkegaard &Perry Laboratories, Inc. (Washington, D.C.)]. Thereafter,phosphotyrosine-containing proteins may be visualized by standardtechniques including colorimetric, chemiluminescent, or fluorescentassays. For example, a chemiluminescent assay may be performed using theECL Western Blotting System from Amersham.

Another cell-based in vitro assay that may be used to assess theactivity of the peptides of the present invention comprises a colonyassay, using murine bone marrow or human peripheral blood cells. Murinebone marrow may be obtained from the femurs of mice, while a sample ofhuman peripheral blood may obtained from a healthy donor. In the case ofperipheral blood, mononuclear cells are first isolated from the blood,for example, by centrifugation through a Ficoll-Hypaque gradient [StemCell Technologies, Inc. (Vancouver, Canada)]. For this assay a nucleatedcell count is performed to establish the number and concentration ofnucleated cells in the original sample. A defined number of cells isplated on methyl cellulose as per manufacturer's instructions [Stem CellTechnologies, Inc. (Vancouver, Canada)]. An experimental group istreated with a test peptide, a positive control group is treated withEPO, and a negative control group receives no treatment. The number ofgrowing colonies for each group is then scored after defined periods ofincubation, generally 10 days and 18 days. An active peptide willpromote colony formation.

Other in vitro biological assays that can be used to demonstrate theactivity of the compounds of the present invention are disclosed inGreenberger, et al. (1983) Proc. Natl. Acad. Sci. USA 80:2931-2935(EPO-dependent hematopoietic progenitor cell line); Quelle andWojchowski (1991) J. Biol. Chem. 266:609-614 (protein tyrosinephosphorylation in B6SUt.EP cells); Dusanter-Fourt, et al. (1992) J.Biol. Chem. 287:10670-10678 (tyrosine phosphorylation of EPO-receptor inhuman EPO-responsive cells); Quelle, et al. (1992) J. Biol. Chem.267:17055-17060 (tyrosine phosphorylation of a cytosolic protein, pp100, in FDC-ER cells); Worthington, et al. (1987) Exp. Hematol. 15:85-92(colorimetric assay for hemoglobin); Kaiho and Miuno (1985) Anal.Biochem. 149:117-120 (detection of hemoglobin with 2,7-diaminofluorene);Patel, et al. (1992) J. Biol. Chem. 267:21300-21302 (expression ofc-myb); Witthuhn, et al. (1993) Cell 74:227-236 (association andtyrosine phosphorylation of JAK2); Leonard, et al. (1993) Blood82:1071-1079 (expression of GATA transcription factors); and Ando, etal. (1993) Proc. Natl. Acad. Sci. USA 90:9571-9575 (regulation of G₁transition by cycling D2 and D3).

An instrument designed by Molecular Devices Corp., known as amicrophysiometer, has been reported to be successfully used formeasurement of the effect of agonists and antagonists on variousreceptors. The basis for this apparatus is the measurement of thealterations in the acidification rate of the extracellular media inresponse to receptor activation.

In Vivo Functional Assays

One in vivo functional assay that may be used to assess the potency of atest peptide is the polycythemic exhypoxic mouse bioassay. For thisassay, mice are subjected to an alternating conditioning cycle forseveral days. In this cycle, the mice alternate between periods ofhypobaric conditions and ambient pressure conditions. Thereafter, themice are maintained at ambient pressure for 2-3 days prior toadministration of test samples. Test peptide samples, or EPO standard inthe case positive control mice, are injected subcutaneously into theconditioned mice. Radiolabeled iron (e.g., Fe⁵⁹) is administered 2 dayslater, and blood samples taken two days after administration ofradiolabeled iron. Hematocrits and radioactivity measurements are thendetermined for each blood sample by standard techniques. Blood samplesfrom mice injected with active test peptides will show greaterradioactivity (due to binding of Fe⁵⁹ by erythrocyte hemoglobin) thanmice that did not receive test peptides or EPO.

Another in vivo functional assay that may be used to assess the potencyof a test peptide is the reticulocyte assay. For this assay, normaluntreated mice are subcutaneously injected on three consecutive dayswith either EPO or test peptide. On the third day, the mice are alsointraperitoneally injected with iron dextran. At day five, blood samplesare collected from the mice. The percent (%) of reticulocytes in theblood is determined by thiazole orange staining and flow cytometeranalysis (retic-count program). In addition, hematocrits are manuallydetermined. The percent of corrected reticulocytes is determined usingthe following formula:

% RETIC_(CORRECTED)=%RETIC_(OBSERVED)×(Hematocrit_(INDIVIDUAL)/Hematocrit_(NORMAL))

Active test compounds will show an increased % RETIC_(CORRECTED) levelrelative to mice that did not receive test peptides or EPO.

Use of EPO-R Agonist Peptides of the Invention

The peptide compounds of the invention are useful in vitro as tools forunderstanding the biological role of EPO, including the evaluation ofthe many factors thought to influence, and be influenced by, theproduction of EPO and the binding of EPO to the EPO-R (e.g., themechanism of EPO/EPO-R signal transduction/receptor activation). Thepresent peptides are also useful in the development of other compoundsthat bind to the EPO-R, because the present compounds provide importantstructure-activity-relationship information that facilitate thatdevelopment.

Moreover, based on their ability to bind to EPO-R, the peptides of thepresent invention can be used as reagents for detecting EPO-R on livingcells; fixed cells; in biological fluids; in tissue homogenates; inpurified, natural biological materials; etc. For example, by labelingsuch peptides, one can identify cells having EPO-R on their surfaces. Inaddition, based on their ability to bind EPO-R, the peptides of thepresent invention can be used in in situ staining, FACS(fluorescence-activated cell sorting) analysis, Western blotting, ELISA(enzyme-linked immunosorbent assay), etc. In addition, based on theirability to bind to EPO-R, the peptides of the present invention can beused in receptor purification, or in purifying cells expressing EPO-R onthe cell surface (or inside permeabilized cells).

The peptides of the invention can also be utilized as commercialreagents for various medical research and diagnostic purposes. Such usescan include but are not limited to: (1) use as a calibration standardfor quantitating the activities of candidate EPO-R agonists in a varietyof functional assays; (2) use as blocking reagents in random peptidescreening, i.e., in looking for new families of EPO-R peptide ligands,the peptides can be used to block recovery of EPO peptides of thepresent invention; (3) use in co-crystallization with EPO-R, i.e.,crystals of the peptides of the present invention bound to the EPO-R maybe formed, enabling determination of receptor/peptide structure by X-raycrystallography; (4) use to measure the capacity of erythrocyteprecursor cells induce globin synthesis and heme complex synthesis, andto increase the number of ferritin receptors, by initiatingdifferentiation; (5) use to maintain the proliferation and growth ofEPO-dependent cell lines, such as the FDCP-1-mEPO-R and the TF-1 celllines; and (6) other research and diagnostic applications wherein theEPO-R is preferably activated or such activation is convenientlycalibrated against a known quantity of an EPO-R agonist, and the like.

In yet another aspect of the present invention, methods of treatment andmanufacture of a medicament are provided. The peptide compounds of theinvention may be administered to warm blooded animals, including humans,to simulate the binding of EPO to the EPO-R in vivo. Thus, the presentinvention encompasses methods for therapeutic treatment of disordersassociated with a deficiency of EPO, which methods compriseadministering a peptide of the invention in amounts sufficient tostimulate the EPO-R and thus, alleviate the symptoms associated with adeficiency of EPO in vivo. For example, the peptides of this inventionwill find use in the treatment of renal insufficiency and/or end-stagerenal failure/dialysis; anemia associated with AIDS; anemia associatedwith chronic inflammatory diseases (for example, rheumatoid arthritisand chronic bowel inflammation) and autoimmune disease; and for boostingthe red blood count of a patient prior to surgery. Other disease states,disorders, and states of hematologic irregularity that may be treated byadministration of the peptides of this invention include:beta-thalassemia; cystic fibrosis; pregnancy and menstrual disorders;early anemia of prematurity; spinal cord injury; space flight; acuteblood loss; aging; and various neoplastic disease states accompanied byabnormal erythropoiesis.

In other embodiments, the peptide compounds of the invention may be usedfor the treatment of disorders which are not characterized by low ordeficient red blood cells, for example as a pretreatment prior totransfusions. In addition, administration of the compounds of thisinvention can result in a decrease in bleeding time and thus, will finduse in the administration to patients prior to surgery or forindications wherein bleeding is expected to occur. In addition, thecompounds of this invention will find use in the activation ofmegakaryoctes.

Since EPO has been shown to have a mitogenic and chemotactic effect onvascular endothelial cells as well as an effect on central cholinergicneurons [see, e.g., Amagnostou, et al. (1990) Proc. Natl. Acad. Sci. USA87:5978-5982 and Konishi, et al. (1993) Brain Res. 609:29-35], thecompounds of this invention will also find use for the treatment of avariety of vascular disorders, such as: promoting wound healing;promoting growth of collateral coronary blood vessels (such as thosethat may occur after myocardial infarction); trauma treatment; andpost-vascular graft treatment. The compounds of this invention will alsofind use for the treatment of a variety of neurological disorders,generally characterized by low absolute levels of acetyl choline or lowrelative levels of acetyl choline as compared to other neuroactivesubstances e.g., neurotransmitters.

Pharmaceutical Compositions

In yet another aspect of the present invention, pharmaceuticalcompositions of the above EPO-R agonist peptide compounds are provided.Conditions alleviated or modulated by the administration of suchcompositions include those indicated above. Such pharmaceuticalcompositions may be for administration by oral, parenteral(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), transdermal (either passively or using iontophoresis orelectroporation), transmucosal (nasal, vaginal, rectal, or sublingual)routes of administration or using bioerodible inserts and can beformulated in dosage forms appropriate for each route of administration.In general, comprehended by the invention are pharmaceuticalcompositions comprising effective amounts of an EPO-R agonist peptide,or derivative products, of the invention together with pharmaceuticallyacceptable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers. Such compositions include diluents of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength;additives such as detergents and solubilizing agents (e.g., Tween 20,Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol); incorporation of thematerial into particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronicacid may also be used. Such compositions may influence the physicalstate, stability, rate of in vivo release, and rate of in vivo clearanceof the present proteins and derivatives. See, e.g., Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa. 18042) pages 1435-1712 which are herein incorporated by reference.The compositions may be prepared in liquid form, or may be in driedpowder (e.g., lyophilized) form.

Oral Delivery

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets, pellets, powders, orgranules. Also, liposomal or proteinoid encapsulation may be used toformulate the present compositions (as, for example, proteinoidmicrospheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation may be used and the liposomes may be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given by Marshall, K.In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter10, 1979, herein incorporated by reference. In general, the formulationwill include the EPO-R agonist peptides (or chemically modified formsthereof) and inert ingredients which allow for protection against thestomach environment, and release of the biologically active material inthe intestine.

Also contemplated for use herein are liquid dosage forms for oraladministration, including pharmaceutically acceptable emulsions,solutions, suspensions, and syrups, which may contain other componentsincluding inert diluents; adjuvants such as wetting agents, emulsifyingand suspending agents; and sweetening, flavoring, and perfuming agents.

The peptides may be chemically modified so that oral delivery of thederivative is efficacious. Generally, the chemical modificationcontemplated is the attachment of at least one moiety to the componentmolecule itself, where said moiety permits (a) inhibition ofproteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecomponent or components and increase in circulation time in the body. Asdiscussed above, PEGylation is a preferred chemical modification forpharmaceutical usage. Other moieties that may be used include: propyleneglycol, copolymers of ethylene glycol and propylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane and poly-1,3,6-tioxocane[see, e.g., Abuchowski and Davis (1981) “Soluble Polymer-EnzymeAdducts,” in Enzymes as Drugs. Hocenberg and Roberts, eds.(Wiley-Interscience: New York, N.Y.) pp. 367-383; and Newmark, et al.(1982) J. Appl. Biochem. 4:185-189].

For oral formulations, the location of release may be the stomach, thesmall intestine (the duodenum, the jejunem, or the ileum), or the largeintestine. One skilled in the art has available formulations which willnot dissolve in the stomach, yet will release the material in theduodenum or elsewhere in the intestine. Preferably, the release willavoid the deleterious effects of the stomach environment, either byprotection of the peptide (or derivative) or by release of the peptide(or derivative) beyond the stomach environment, such as in theintestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic (i.e. powder), for liquid forms a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The peptide (or derivative) can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs, or even as tablets.These therapeutics could be prepared by compression.

Colorants and/or flavoring agents may also be included. For example, thepeptide (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the peptide (or derivative)with an inert material. These diluents could include carbohydrates,especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose,modified dextrans and starch. Certain inorganic salts may be also beused as fillers including calcium triphosphate, magnesium carbonate andsodium chloride. Some commercially available diluents are Fast-Flo,Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. The disintegrants may also be insoluble cationicexchange resins. Powdered gums may be used as disintegrants and asbinders. and can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the peptide (or derivative) agent togetherto form a hard tablet and include materials from natural products suchas acacia, tragacanth, starch and gelatin. Others include methylcellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC).Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC)could both be used in alcoholic solutions to granulate the peptide (orderivative).

An antifrictional agent may be included in the formulation of thepeptide (or derivative) to prevent sticking during the formulationprocess. Lubricants may be used as a layer between the peptide (orderivative) and the die wall, and these can include but are not limitedto; stearic acid including its magnesium and calcium salts,polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils andwaxes. Soluble lubricants may also be used such as sodium laurylsulfate, magnesium lauryl sulfate, polyethylene glycol of variousmolecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the peptide (or derivative) into the aqueousenvironment a surfactant might be added as a wetting agent. Surfactantsmay include anionic detergents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergentsmight be used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, sucrosefatty acid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives which potentially enhance uptake of the peptide (orderivative) are for instance the fatty acids oleic acid, linoleic acidand linolenic acid.

Controlled release oral formulations may be desirable. The peptide (orderivative) could be incorporated into an inert matrix which permitsrelease by either diffusion or leaching mechanisms, e.g., gums. Slowlydegenerating matrices may also be incorporated into the formulation.Some enteric coatings also have a delayed release effect. Another formof a controlled release is by a method based on the Oros therapeuticsystem (Alza Corp.), i.e. the drug is enclosed in a semipermeablemembrane which allows water to enter and push drug out through a singlesmall opening due to osmotic effects.

Other coatings may be used for the formulation. These include a varietyof sugars which could be applied in a coating pan. The peptide (orderivative) could also be given in a film coated tablet and thematerials used in this instance are divided into 2 groups. The first arethe nonenteric materials and include methyl cellulose, ethyl cellulose,hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropylcellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methylcellulose, providone and the polyethylene glycols. The second groupconsists of the enteric materials that are commonly esters of phthalicacid.

A mix of materials might be used to provide the optimum film coating.Film coating may be carried out in a pan coater or in a fluidized bed orby compression coating.

Parenteral Delivery

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured using sterile water, or some other sterile injectablemedium, immediately before use.

Rectal or Vaginal Delivery

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients well known in the art.

Pulmonary Delivery

Also contemplated herein is pulmonary delivery of the EPO-R agonistpeptides (or derivatives thereof). The peptide (or derivative) isdelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream [see, e.g., Adjei, et al.(1990) Pharmaceutical Research 7:565-569; Adjei, et al. (1990) Int. J.Pharmaceutics 63:135-144 (leuprolide acetate); Braquet, et al. (1989) J.Cardiovascular Pharmacology 13(sup5):143-146 (endothelin-1); Hubbard, etal. (1989) Annals of Internal Medicine, Vol. III, pp. 206-212(α1-antitrypsin); Smith, et al. (1989) J. Clin. Invest. 84:1145-1146(α-1-proteinase); Oswein, et al. (1990) “Aerosolization of Proteins”,Proceedings of Symposium on Respiratory Drug Delivery II Keystone,Colorado (recombinant human growth hormone); Debs, et al. (1988) J.Immunol. 140:3482-3488 (interferon-γ and tumor necrosis factor α); andU.S. Pat. No. 5,284,656 to Platz, et al. (granulocyte colony stimulatingfactor). A method and composition for pulmonary delivery of drugs forsystemic effect is described in U.S. Pat. No. 5,451,569 to Wong, et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer(Marquest Medical Products, Englewood, Colo.); the Ventolin metered doseinhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhalerpowder inhaler (Fisons Corp., Bedford, Mass.).

All such devices require the use of formulations suitable for thedispensing of peptide (or derivative). Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to the usual diluents,adjuvants and/or carriers useful in therapy. Also, the use of liposomes,microcapsules or microspheres, inclusion complexes, or other types ofcarriers is contemplated. Chemically modified peptides may also beprepared in different formulations depending on the type of chemicalmodification or the type of device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise peptide (or derivative) dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of thepeptide (or derivative) caused by atomization of the solution in formingthe aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the peptide (or derivative)suspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing peptide (or derivative) and mayalso include a bulking agent, such as lactose, sorbitol, sucrose, ormannitol in amounts which facilitate dispersal of the powder from thedevice, e.g., 50 to 90% by weight of the formulation. The peptide (orderivative) should most advantageously be prepared in particulate formwith an average particle size of less than 10 mm (or microns), mostpreferably 0.5 to 5 mm, for most effective delivery to the distal lung.

Nasal Delivery

Nasal delivery of the EPO-R agonist peptides (or derivatives) is alsocontemplated. Nasal delivery allows the passage of the peptide to theblood stream directly after administering the therapeutic product to thenose, without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran.

Dosages

For all of the peptide compounds, as further studies are conducted,information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age, and generalhealth of the recipient, will be able to ascertain proper dosing. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment desired.Generally dosage levels of 0.001 to 10 mg/kg of body weight daily areadministered to mammals. Generally, for intravenous injection orinfusion dosage may be lower. The dosing schedule may vary, depending onthe circulation half-life, and the formulation used.

The peptides of the present invention (or their derivatives) may beadministered in conjunction with one or more additional activeingredients or pharmaceutical compositions.

EXAMPLES

The present invention is next described by means of the followingexamples. However, the use of these and other examples anywhere in thespecification is illustrative only, and in no way limits the scope andmeaning of the invention or of any exemplified form. Likewise, theinvention is not limited to any particular preferred embodimentsdescribed herein. Indeed, many modifications and variations of theinvention may be apparent to those skilled in the art upon reading thisspecification, and can be made without departing from its spirit andscope. The invention is therefore to be limited only by the terms of theappended claims, along with the full scope of equivalents to which theclaims are entitled.

Example 1 Synthesis of EPO-R Agonist Peptides 1. Peptide MonomerSynthesis

Various peptide monomers of the invention were synthesized using theMerrifield solid phase synthesis technique [see, Stewart and Young.Solid Phase Peptide Synthesis, 2^(nd) edition (Pierce Chemical,Rockford, Ill.) 1984] on an Applied Biosystems 433A automatedinstrument. The resin used was PAL (Milligen/Biosearch), which iscross-linked polystyrene with5-(4′-Fmoc-aminomethyl-3,5′-dimethoxyphenoxy) valeric acid. Use of PALresin results in a carboxyl terminal amide function upon cleavage of thepeptide from the resin. Primary amine protection on amino acids wasachieved with Fmoc, and side chain protection groups were t-butyl forserine, threonine, and tyrosine hydroxyls; trityl for glutamine andasparagine amides; Trt or Acm for cysteine; and Pmc(2,2,5,7,8-pentamethylchroman sulfonate) for the arginine guanidinogroup. Each coupling was performed for either 1 hr or 2 hr with BOP(benzotriazolyl N-oxtrisdimethylaminophosphonium hexafluorophosphate)and HOBt (1-hydroxybenztriazole).

For the synthesis of peptides with an amidated carboxy terminus, thefully assembled peptide was cleaved with a mixture of 90%trifluoroacetic acid, 5% ethanedithiol, and 5% water, initially at 4° C.and gradually increasing to room temperature over 1.5 hr. Thedeprotected product was filtered from the resin and precipitated withdiethyl ether. After thorough drying the product was purified by C18reverse phase high performance liquid chromatography with a gradient ofacetonitrile/water in 0.1% trifluoroacetic acid.

2. Peptide Dimer Synthesis

Various peptide dimers of the invention were synthesized directly onto alysine linker in a variation of the solid phase technique.

For simultaneous synthesis of the two peptide chains, Fmoc-Lys-Fmoc wascoupled to a PAL resin (Milligen/Biosearch), thereby providing aninitial lysine residue to serve as the linker between the two chains tobe synthesized. The Fmoc protecting groups were removed with mild base(20% piperidine in DMF), and the peptide chains were synthesized usingthe resulting free amino groups as starting points. Peptide chainsynthesis was performed using the solid phase synthesis techniquedescribed above. Trt was used to protect all cysteine residues.Following dimer deprotection, cleavage from the resin, and purification,oxidation of the cysteine residues was performed by incubating thedeprotected dimer in 100% DMSO for 2-3 days at 5° C. to 25° C. Thisoxidation reaction yielded predominantly (>75%) dimers with twointramolecular disulfide bonds.

For sequential synthesis of the two peptide chains, Fmoc-Lys-Alloc wascoupled to a PAL resin (Milligen/Biosearch), thereby providing aninitial lysine residue to serve as the linker between the two chains tobe synthesized. The Fmoc protecting group was removed with mild base(20% piperidine in DMF). The first peptide chain was then synthesizedusing the resulting free amino group as a starting point. Peptidesynthesis was performed using the solid phase technique described above.The two cysteine residues of the first chain were protected with Trt.Following synthesis of the first peptide chain, the Alloc group wasremoved from the support-bound lysine linker with Pd[P(C₆H₅)₃]₄,4-methyl morpholine, and chloroform. The second peptide chain was thensynthesized on this second free amino group. The two cysteine residuesof the second chain were protected with Acm. An intramolecular disulfidebond was formed in the first peptide chain by removing the Trtprotecting groups using trifluoroacetic acid, followed by oxidation bystirring in 20% DMSO overnight. An intramolecular disulfide bond wasthen formed in the second peptide chain by simultaneously removing theAcm protecting groups and oxidizing the deprotected cysteine residuesusing iodine, methanol, and thalium trifluoroacetate.

3. Attachment of Spacers

Where the spacer was an amino acid (e.g., glycine or lysine as inAF35462 and AF35464, respectively), the spacer was incorporated into thepeptide during solid phase peptide synthesis. In this case, the spaceramino acid was coupled to the PAL resin, and its free amino group servedas the basis for the attachment of another spacer amino acid, or of thelysine linker. Following the attachment of the lysine linker, dimericpeptides were synthesized as described above.

4. Synthesis of Exemplary Peptide Dimers

Exemplary embodiments of these synthesis techniques are outlined below.In one example, the synthesis of a peptide dimer linked via a C-terminallysine amide is described. In another example, the synthesis of apeptide dimer linked via a C-terminal lysine, and containing a spacermolecule attached to the linking lysine, is described.

Synthesis of a Peptide Dimer Linked Via a C-Terminal Lysine Amide:

Step 1—Formation of TentaGel-Rink-Lys:

TentaGel-Rink resin (0.18 mml/g from Rapp Polymere, Germany) was treatedwith a activated solution of Fmoc-Lys(Fmoc)-OH (prepared from 5 eq. ofamino acid and 5 eq. of HATU dissolved at 0.5 M in DMF, followed by theaddition of 10 eq. of DIEA) and allowed to gently shake 14 h. The resinwas washed (DMF, THF, DCM, MeOH) and dried to yield the protected resin.Residual amine groups were capped by treating the resin with a solutionof 10% acetic anhydride, 20% pyridine in DCM for 20 minutes, followed bywashing as above. The Fmoc groups were removed by gently shaking theresin in 30% piperidine in DMF for 20 minutes, followed by washing (DMF,THF, DCM, MeOH) and drying.

Step 2—Formation of TentaGel-Rink-Lys(Peptide)₂:

The resin from Step 1 was subjected to repeated cycles of Fmoc-aminoacid couplings with HBTU/HOBt activation and Fmoc removal withpiperidine to build both peptide chains simultaneously. This wasconvenently carried out on a ABI 433 automated peptide synthesizeravailable from Applied Biosystems, Inc. After the final Fmoc removal,the terminal amine groups were acylated with acetic anhydride (10 eq.)and DIEA (20 eq.) in DMF for 20 minutes, follwed by washing as above.

Step 3—Cleavage from Resin:

The resin from above is suspended in to a solution of TFA (82.5%),phenol (5%), ethanedithiol (2.5%), water (5%), and thioanisole (5%) for3 h at room temperature. Alternative cleavage cocktails such as TFA(95%), water (2.5%), and triisopropylsilane (2.5%) can also be used. TheTFA solution is cooled to 5° C. and poured into Et₂O to precipitate thepeptide. Filtration and drying under reduced pressure gave the desiredpeptide. Purification via preparative HPLC with a C18 column affords thepure peptide.

Synthesis of a Peptide Dimer Linked Via a C-Terminal Lysine Amide andContaining a Spacer Molecule

Step 1—Synthesis of Cbz-TAP:

A solution containing the commercially available diamine (“TAP” fromAldrich Chemical Co.) (10 g, 67.47 mmol) in anhydrous DCM (100 ml) wascooled to 0° C. A solution of benzyl chloroformate (4.82 ml, 33.7 mmol)in anhydrous DCM (50 ml) was added slowly through a dropping funnel overa period of 6-7 h, maintaining the temperature of the reaction mixtureat 0° C. throughout, then allowed to warm to room temperature (˜25° C.).After a further 16 h, the DCM was removed under vacuum and the residuepartitioned between 3N HCl and ether. The aqueous layers were collectedand neutralized with 50% aq. NaOH to pH8-9 and extracted with ethylacetate. The ethyl acetate layer was dried over anhydrous Na₂SO₄, thenconcentrated under vacuum to provide the crude mono-Cbz-TAP (5 g, about50% yield). This compound was used for the next reaction without anyfurther purification.

Step 2—Synthesis of Cbz-TAP-Boc:

To a vigorously stirred suspension of the Cbz-TAP (5 g, 17.7 mmol) inhexane (25 ml) was added Boc₂O (3.86 g, 17.7 mmol) and stirringcontinued at RT overnight. The reaction mixture was diluted with DCM (25ml) and washed with 10% aq. citric acid (2×), water (2×) and brine. Theorganic layer was dried over anhydrous Na₂SO₄ and concentrated undervacuum. The crude product (yield 5 g) was used directly in the nextreaction.

Step 3—Synthesis of Boc-TAP:

The crude product from the previous reaction was dissolved in methanol(25 ml) and hydrogenated in presence of 5% Pd on Carbon (5% w/W) underballoon pressure for 16 hrs. The mixture was filtered, washed withmethanol and the filtrate concentrated in vacuo to provide the crudeH-TAP-Boc product (yield 3.7 g). The overall approximate yield ofBoc-TAP after Steps 1-3 was 44% (calculated based on the amount ofCbz-Cl used.)

Step 4—Synthesis of TentaGel-Linker:

TentaGel bromide (2.5 g, 0.48 mmol/g, from Rapp Polymere, Germany),phenolic linker (5 equivalent, and K₂CO₃ (5 equivalent) were heated in20 mL of DMFto 70° C. for 14 h. After cooling to room temperature, theresin was washed (0.1 N HCl, water, ACN, DMF, MeOH) and dried to give anamber-colored resin.

Step 5—Synthesis of TentaGel-Linker-TAP(Boc):

2.5 gms of the resin from above and H-TAP-Boc (1.5 gms, 5 eq.) andglacial AcOH (34 μl, 5 eq.) was taken in a mixture of 1:1 MeOH-THF andshaken overnight. A 1 M solution of sodium cyanoborohydride (5 eq) inTHF was added to this and shaken for another 7 hrs. The resin wasfiltered washed (DMF, THF, 0.1 N HCl, water, MeOH) and dried. A smallamount of the resin was benzoylated with Bz-Cl and DIEA in DCM andcleaved with 70% TFA-DCM and checked by LCMS and HPLC.

Step 6—Synthesis of TentaGel-Linker-TAP-Lys:

The resin from above was treated with a activated solution ofFmoc-Lys(Fmoc)-OH (prepared from 5 eq. of amino acid and 5 eq. of HATUdissolved at 0.5 M in DMF, followed by the addition of 10 eq. of DIEA)and allowed to gently shake 14 h. The resin was washed (DMF, THF, DCM,MeOH) and dried to yield the protected resin. Residual amine groups werecapped by treating the resin with a solution of 10% acetic anhydride,20% pyridine in DCM for 20 minutes, followed by washing as above. TheFmoc groups are removed by gently shaking the resin in 30% piperideinein DMF for 20 minutes, followed by washing (DMF, THF, DCM, MeOH) anddrying.

Step 7—Synthesis of TentaGel-Linker-TAP-Lys(Peptide)₂:

The resin from above was subjected to repeated cycles of Fmoc-amino acidcouplings with HBTU/HOBt activation and Fmoc removal with piperidine tobuild both peptide chains simultaneously. This was conveniently carriedout on a ABI 433 automated peptide synthesizer available from AppliedBiosystems, Inc. After the final Fmoc removal, the terminal amine groupswere acylated with acetic anhydride (10 eq.) and DIEA (20 eq.) in DMFfor 20 minutes, followed by washing as above.

Step 8—Cleavage from Resin:

The resin from above was suspended in a solution of TFA (82.5%), phenol(5%), ethanedithiol (2.5%), water (5%), and thioanisole (5%) for 3 h atroom temperature. Alternative cleavage cocktails such as TFA (95%),water (2.5%), and triisopropylsilane (2.5%) can also be used. The TFAsolution was cooled to 5° C. and poured into Et₂O to precipitate thepeptide. Filtration and drying under reduced pressure gave the desiredpeptide. Purification via preparative HPLC with a C18 column affordedthe pure peptide.

5. Oxidation of Peptides to Form Intramolecular Disulfide Bonds

The peptide dimer was dissolved in 20% DMSO/water (1 mg dry weightpeptide/mL) and allowed to stand at room temperature for 36 h. Thepeptide was purified by loading the reaction mixture onto a C18 HPLCcolumn (Waters Delta-Pak C18, 15 micron particle size, 300 angstrom poresize, 40 mm×200 mm length), followed by a linear ACN/water/0.01% TFAgradiant from 5 to 95% ACN over 40 minutes. Lypholization of thefractions containing the desired peptide affords the product as a fluffywhite solid. For example, in the case of AF35525, this reaction may beillustrated schematically as follows:

6. PEGylation of Peptides

PEGylation of the peptides of the invention was carried out usingseveral different techniques.

PEGylation of a Terminal —NH₂ Group:

The peptide dimer was mixed with 1.5 eq. (mole basis) of activated PEGspecies (mPEG-NPC from NOF Corp. Japan) in dry DMF to afford a clearsolution.

After 5 minutes 4 eq of DIEA was added to above solution. The mixturewas stirred at ambient temperature 14 h, followed by purification withC18 reverse phase HPLC. The structure of PEGylated peptide was confirmedby MALDI mass. The purified peptide was also subjected to purificationvia cation ion exchange chromatography as outlined below. For example,in the case of AF35593, the monoPEGylation of the terminal —NH₂ group ofthe spacer moiety may be illustrated schematically as follows:

DiPEGylation of the N-Termini of a Peptide Dimer:

The peptide dimer is mixed with 2.5 eq. (mole basis) of activated PEGspecies (mPEG-NPC from NOF Corp. Japan) in dry DMF to afford a clearsolution. After 5 minutes 4 eq of DIEA is added to above solution. Themixture is stirred at ambient temperature 14 h, followed by purificationwith C18 reverse phase HPLC. The purified peptide is also subjected topurification via cation ion exchange chromatography as outlined below.For example, in the case of AF35083, this reaction may be illustratedschematically as follows:

Peptide Dimerization Via PEGylation of N-Termini:

The peptide (2.5 eq.) and PEG-(SPA-NHS)₂ (1 eq. from Shearwater Corp,USA.) were dissolved in dry DMF at 0.25M to afford a clear solution.After 5 minutes 10 eq of DIEA is added to above solution. The mixture isstirred at ambient temperature 2 h, followed by purification with C18reverse phase HPLC. The purified peptide is also subjected topurification via cation ion exchange chromatography as outlined below.For example, in the case of AF33131, this reaction may be illustratedschematically as follows:

Peptide Dimerization Via PEGylation of C-Termini:

The peptide (2.5 eq.) and PEG-(SPA-NHS)₂ (1 eq. from Shearwater Corp,USA.) were dissolved in dry DMF at 0.25M to afford a clear solution.After 5 minutes 10 eq of DMA is added to above solution. The mixture isstirred at ambient temperature 2 h, followed by purification with C18reverse phase HPLC. The purified peptide is also subjected topurification via cation ion exchange chromatography as outlined below.For example, this reaction may be summarized as follows:

7. Ion Exchange Purification of Peptides.

Several exchange supports were surveyed for their ability to separatethe above peptide-PEG conjugate from unreacted (or hydrolyzed) PEG, inaddition to their ability to retain the starting dimeric peptides. Theion exchange resin (2-3 g) was loaded into a 1 cm column, followed byconversion to the sodium form (0.2 N NaOH loaded onto column untilelutant was pH 14, ca. 5 column volumes), and than to the hydrogen form(eluted with either 0.1 N HCl or 0.1 M HOAc until elutant matched loadpH, ca. 5 column volumes), followed by washing with 25% ACN/water untilpH 6. Either the peptide prior to conjugation or the peptide-PEGconjugate was dissolved in 25% ACN/water (10 mg/mL) and the pH adjustedto <3 with TFA, then loaded on the column. After washing with 2-3 columnvolumes of 25% ACN/water and collecting 5 mL fractions, the peptide wasreleased from the column by elution with 0.1 M NH₄OAc in 25% ACN/water,again collecting 5 mL fractions. Analysis via HPLC revealed whichfractions contained the desired peptide. Analysis with an EvaporativeLight-Scattering Detector (ELSD) indicated that when the peptide wasretained on the column and was eluted with the NH₄OAc solution(generally between fractions 4 and 10), no non-conjugated PEG wasobserved as a contaminant. When the peptide eluted in the initial washbuffer (generally the first 2 fractions), no separation of desiredPEG-conjugate and excess PEG was observed.

The following columns successfully retained both the peptide and thepeptide-PEG conjugate, and successfully purified the peptide-PEGconjugate from the unconjugates peptide:

TABLE 1 Ion Exhange Resins Support Source Mono S HR 5/5 strong cationAmersham Biosciences exchange pre-loaded column SE53 Cellulose,microgranular Whatman strong cation exchange support SP Sepharose FastFlow strong Amersham Biosciences cation exchange support

Example 2 In Vitro Activity Assays

This example describes various in vitro assays that are useful inevaluating the activity and potency of EPO-R agonist peptides of theinvention. The results for these assays demonstrate that the novelpeptides of this invention bind to EPO-R and activate EPO-R signaling.Moreover, the results for these assays show that the novel peptidecompositions exhibit a surprising increase in EPO-R binding affinity andbiological activity compared to EPO mimetic peptides that have beenpreviously described.

EPO-R agonist peptide monomers and dimers are prepared according to themethods provided in Example 1. The potency of these peptide monomers anddimers is evaluated using a series of in vitro activity assays,including: a reporter assay, a proliferation assay, a competitivebinding assay, and a C/BFU-e assay. These four assays are described infurther detail below.

The results of these in vitro activity assays are summarized in Table 2(for peptide monomers) and Table 3 (for peptide dimers). These tablesprovide the compound designation and structure for each tested peptide,as well as the experimental results for each of these four assays. Theseresults demonstrate the dramatically enhanced potency of the novelpeptides of the invention.

1. Reporter Assay

This assay is based upon a on a murine pre-B-cell line derived reportercell, Baf3/EpoR/GCSFR fos/lux. This reporter cell line expresses achimeric receptor comprising the extra-cellular portion of the human EPOreceptor to the intra-cellular portion of the human GCSF receptor. Thiscell line is further transfected with a fos promoter-driven luciferasereporter gene construct. Activation of this chimeric receptor throughaddition of erythropoietic agent results in the expression of theluciferase reporter gene, and therefore the production of light uponaddition of the luciferase substrate luciferin. Thus, the level of EPO-Ractivation in such cells may be quantitated via measurement ofluciferase activity.

The Baf3/EpoR/GCSFR fos/lux cells are cultured in DMEM/F12 medium(Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone), 10%WEHI-3 supernatant (the supernatant from a culture of WEHI-3 cells, ATCC# TIB-68), and penicillin/streptomycin. Approximately 18 h before theassay, cells are starved by transferring them to DMEM/F12 mediumsupplemented with 10% FBS and 0.1% WEHI-3 supernatant. On the day ofassay, cells are washed once with DMEM/F12 medium supplemented with 10%FBS (no WEHI-3 supernatant), then 1×10⁶ cells/mL are cultured in thepresence of a known concentration of test peptide, or with EPO(R & DSystems Inc., Minneapolis, Minn.) as a positive control, in DMEM/F12medium supplemented with 10% FBS (no WEHI-3 supernatant). Serialdilutions of the test peptide are concurrently tested in this assay.Assay plates are incubated for 4 h at 37° C. in a 5% CO₂ atmosphere,after which luciferin (Steady-Glo; Promega, Madison, Wi) is added toeach well. Following a 5-minute incubation, light emission is measuredon a Packard Topcount Luminometer (Packard Instrument Co., DownersGrove, Ill.). Light counts are plotted relative to test peptideconcentration and analysed using Graph Pad software. The concentrationof test peptide that results in a half-maximal emission of light isrecorded as the EC50 [See Tables 2 and 3: Reporter EC50].

2. Proliferation Assay

This assay is based upon a murine pre-B-cell line, Baf3, transfected toexpress human EPO-R. Proliferation of the resulting cell line,BaF3/Ga14/Elk/EPOR, is dependent on EPO-R activation. The degree of cellproliferation is quantitated using MTT, where the signal in the MTTassay is proportional to the number of viable cells.

The BaF3/Ga14/Elk/EPOR cells are cultured in spinner flasks in DMEM/F12medium (Gibco) supplemented with 10% FBS (Hyclone) and 2% WEHI-3supernatant (ATCC # TIB-68). Cultured cells are starved overnight, in aspinner flask at a cell density of 1×10⁶ cells/ml, in DMEM/F12 mediumsupplemented with 10% FBS and 0.1% WEHI-3 supernatant. The starved cellsare then washed twice with Dulbecco's PBS (Gibco), and resuspended to adensity of 1×10⁶ cells/ml in DMEM/F12 supplemented with 10% FBS (noWEHI-3 supernatant). 500, aliquots (50,000 cells) of the cell suspensionare then plated, in triplicate, in 96 well assay plates. 504 aliquots ofdilution series of test EPO mimetic peptides, or 50 μL EPO(R & D SystemsInc., Minneapolis, Minn.) or Aranesp™ (darbepoeitin alpha, an ERO—Ragonist commerically available from Amgen) in DMEM/F12 mediasupplemented with 10% FBS (no WEHI-3 supernatant I) are added to the 96well assay plates (final well volume of 1004). For example, 12 differentdilutions may be tested where the final concentration of test peptide(or control EPO peptide) ranges from 810 μM to 0.0045 μM. The platedcells are then incubated for 48 h at 37° C. Next, 14 μL of MIT (RocheDiagnostics) is added to each culture dish well, and then allowed toincubate for 4 h. The reaction is then stopped by adding 10% SDS+0.01NHCl. The plates are then incubated overnight at 37° C. Absorbance ofeach well at a wavelength of 595 nm is then measured byspectrophotometry. Plots of the absorbance readings versus test peptideconcentration are constructed and the EC50 calculated using Graph Padsoftware. The concentration of test peptide that results in ahalf-maximal absorbance is recorded as the EC50 [See Table 3:Proliferation EC50].

3. Competitive Binding Assay

Competitive binding calculations are made using an assay in which alight signal is generated as a function of the proximity of two beads: astreptavidin donor bead bearing a biotinylated EPO-R-binding peptidetracer and an acceptor bead to which is bound EPO-R. Light is generatedby non-radiative energy transfer, during which a singlet oxygen isreleased from a first bead upon illumination, and contact with thereleased singlet oxygen causes the second bead to emit light. These beadsets are commercially available (Packard). Bead proximity is generatedby the binding of the EPO-R-binding peptide tracer to the EPO-R. A testpeptide that competes with the EPO-R-binding peptide tracer for bindingto EPO-R will prevent this binding, causing a decrease in lightemission.

In more detail the method is as follows: Add 4 μL of serial dilutions ofthe test EPO-R agonist peptide, or positive or negative controls, towells of a 384 well plate. Thereafter, add 2 μL/well of receptor/beadcocktail. Receptor bead cocktail consists of: 15 μL of 5 mg/mlstreptavidin donor beads (Packard), 15 μL of 5 mg/ml monoclonal antibodyab179 (this antibody recognizes the portion of the human placentalalkaline phosphatase protein contained in the recombinant EPO-R),protein A-coated acceptor beads (protein A will bind to the ab179antibody; Packard), 112.5 μL of a 1:6.6 dilution of recombinant EPO-R(produced in Chinese Hamster Ovary cells as a fusion protein to aportion of the human placental alkaline phosphatase protein whichcontains the ab179 target epitope) and 607.54 of Alphaquest buffer (40mM HEPES, pH 7.4; 1 mM MgCl₂; 0.1% BSA, 0.05% Tween 20). Tap to mix. Add2 μL/well of the biotinylated EPO-R-binding peptide tracer, AF33068 (30nM final concentration). AF33068, an EPO-R binding peptide (see Table 3“Reporter EC50 (pM)”), is made according to the methods described inExample 1.

Centrifuge 1 min to mix. Seal plate with Packard Top Seal and wrap infoil. Incubate overnight at room temperature. After 18 hours read lightemission using an AlphaQuest reader (Packard). Plot light emission vsconcentration of peptide and analyse with Graph Pad or Excel.

The concentration of test peptide that results in a 50% decrease inlight emission, relative to that observed without test peptide, isrecorded as the IC50 [See Tables 2 and 3: AQ IC50].

4. C/BFU-e Assay

EPO-R signaling stimulates the differentiation of bone marrow stem cellsinto proliferating red blood cell presursors. This assay measures theability of test peptides to stimulate the proliferation anddifferentiation of red blood cell precursors from primary human bonemarrow pluripotent stem cells.

For this assay, serial dilutions of test peptide are made in IMDM medium(Gibco) supplemented with 10% FBS (Hyclone). These serial dilutions, orpositive control EPO peptide, are then added to methylcellulose to givea final volume of 1.5 mL. The methylcellulose and peptide mixture isthen vortexed thoroughly. Aliquots (100,000 cells/mL) of human, bonemarrow derived CD34+ cells (Poietics/Cambrex) are thawed. The thawedcells are gently added to 0.1 mL of 1 mg/ml DNAse (Stem Cells) in a 50mL tube. Next, 40-50 mL IMDM medium is added gently to cells: the mediumis added drop by drop along the side of the 50 mL tube for the first 10mL, and then the remaining volume of medium is slowly dispensed alongthe side of the tube. The cells are then spun at 900 rpm for 20 min, andthe media removed carefully by gentle aspiration. The cells areresuspended in 1 ml of IMDM medium and the cell density per mL iscounted on hemacytometer slide (10 μL aliquot of cell suspension onslide, and cell density is the average count×10,000 cells/ml). The cellsare then diluted in IMDM medium to a cell density of 15,000 cells/mL. A100 μL of diluted cells is then added to each 1.5 mL methyl celluloseplus peptide sample (final cell concentration in assay media is 1000cells/mL), and the mixture is vortexed. Allow the bubbles in the mixtureto disappear, and then aspirate 1 mL using blunt-end needle. Add 0.25 mLaspirated mixture from each sample into each of 4 wells of a 24-wellplate (Falcon brand). Incubate the plated mixtures at 37° C. under 5%CO₂ in a humid incubator for 14 days. Score for the presence oferythroid colonies using a phase microscope (5×−10× objective, finalmagnification of 100×). The concentration of test peptide at which thenumber of formed colonies is 90% of maximum, relative to that observedwith the EPO positive control, is recorded as the EC90 [See Table 3:C/BFU-e EC90].

5. Radioligand Competitive Binding Assay

An alternative radioligand competition binding assay can also be used tomeasure IC₅₀ values for peptides of the present invention. This assaymeasures binding of ¹²⁵I-EPO to EPOr. The assay may be performedaccording to the following exemplary protocol:

A. Materials

Recombinant Identification: Recombinant Human EPO R/Fc Chimera Human EPOSupplier: R&D Systems (Minneapolis, MN, US) R/Fc Chimera Catalog number:963-ER Lot number: EOK033071 Storage: 4° C. Iodinated Identification:(3[¹²⁵I]iodotyrosyl)Erythropoietin, recombinant human recombinant, highspecific activity, human 370 kBq, 10 μCi Erythropoietin Supplier:Amersham Biosciences (Piscataway, NJ, US) Catalog number: IM219-10 μCiLot number: Storage: 4° C. Protein-G Identification: Protein-G Sepharose4 Fast Flow Sepharose Supplier: Amersham Biosciences (Piscataway, NJ,US) Catalog number 17-0618-01 Lot number: Storage: 4° C. Assay BufferPhosphate Buffered Saline (PBS), pH 7.4, containing 0.1% Bovine SerumAlbumin and 0.1% Sodium Azide Storage: 4° C.

B. Determination of Appropriate Receptor Concentration.

One 50 μg vial of lyophilized recombinant EPOr extracellular domainfused to the Fc portion of human IgG1 is reconstituted in 1 mL of assaybuffer. To determine the correct amount of receptor to use in the assay,100 μL serial dilutions of this receptor preparation are combined withapproximately 20,000 cpm in 200 μL of iodinated recombinant humanErythropoietin (¹²⁵I-EPO) in 12×75 mm polypropylene test tubes. Tubesare capped and mixed gently at 4° C. overnight on a LabQuake rotatingshaker.

The next day, 50 μL of a 50% slurry of Protein-G Sepharose is added toeach tube. Tubes are then incubated for 2 hours at 4° C., mixing gently.The tubes are then centrifuged for 15 min at 4000 RPM (3297×G) to pelletthe protein-G sepharose. The supernatants are carefully removed anddiscarded. After washing 3 times with 1 mL of 4° C. assay buffer, thepellets are counted in a Wallac Wizard gamma counter. Results were thenanalyzed and the dilution required to reach 50% of the maximum bindingvalue was calculated.

C. IC₅₀ Determination for Peptide

To determine the IC₅₀ of a peptide of the present invention, 100 μLserial dilutions of the peptide are combined with 100 μL of recombinanterythropoietin receptor (100 pg/tube) in 12×75 mm polypropylene testtubes. Then 100 μL of iodinated recombinant human Erythropoietin(¹²⁵I-EPO) is added to each tube and the tubes were capped and mixedgently at 4° C. overnight.

The next day, bound ¹²⁵I-EPO is quantitated as described above. Theresults are analyzed and the IC₅₀ value calculated using Graphpad Prismversion 4.0, from GraphPad Software, Inc. (San Diego, Calif.) The assayis preferably repeated 2 or more times for each peptide whose IC₅₀ valueis measured by this procedure, for a total of 3 replicate IC₅₀determinations.

6. Discussion

The in vitro reporter assay results for peptide monomers of the presentinvention were directly compared with those for related peptidesequences previously disclosed (see AF31552 and AF31748 in Table 2):namely,

SEQ ID NO: 32 GGLYACHMGPMTVCQPLRG and SEQ ID NO: 33GGLYACHMGPMT(1-nal)VCQPLRG.

These results demonstrate the dramatically improved potency of the novelpeptide monomers of the invention, as the novel peptide dimers were 3 to7.5 times as potent as the previously disclosed peptide monomers in thereporter assay. These novel peptide monomers were then used to preparenovel peptide dimers of even greater potency and activity.

TABLE 2 In vitro reporter assay for peptide monomers Compound Reporterdesignation Peptide monomer EC50 (nM) AF31552

100 AF31748

40 AF33128

13 AF36729

13.3

TABLE 3 In vitro activity assays for peptide dimers ReporterProliferation AQ C/BFU-e EC90 Compound designation Peptide dimer EC50(pM) EC50 (pM) IC50 (nM) (nM) AF33065

300 — — — AF34602

727 — — — AF34395

100 — — — AF34601

100 — — — AF32579

47 142 1.7 3 AF33068

170 — — — AF33131

158 — 18 — AF34351

50 — — — AF34350

267 — 16 — AF34753

73 — — 1.2 AF34757

110 — — 2.7 AF35062

91 — — 1.6 AF35218

27 — — — AF35462

194 — — — AF35464

181 — — — AF33197

80 — — — AF34994

27 170 — 3.7 AF35083

92 — — — AF35525

57 57 — 3 AF35526

900 800 — 13 AF35563

135 47 — 3 AF35575

127 57,000 — 1.1 AF35592

67 40 — 1.3 AF35593

76 32 — 1.5 AF35594

54 40 — 2.2 AF35219

403 — — — AF32876

100 — — — AF32881

100 — — 11 AF35179

57 — — 3.7 AF35180

303 — — — AF35463

125 — — — AF35090

35 190 — 3.7 AF35148

3600 — — — AF35149

43 57 — 2 AF35168

1800 — — — AF35361

77 — — — AF35595

65 42 — 1.5 AF35564

887 270 — 4.5

Example 3 In Vivo Activity Assays

This example describes various in vivo assays that are useful inevaluating the activity and potency of EPO-R agonist peptides of theinvention. EPO-R agonist peptide monomers and dimers are preparedaccording to the methods provided in Example 1. The in vivo activity ofthese peptide monomers and dimers is evaluated using a series assays,including a polycythemic exhypoxic mouse bioassay and a reticulocyteassay. These two assays are described in further detail below.

1. Polycythemic Exhypoxic Mouse Bioassay

Test peptides are assayed for in vivo activity in the polycythemicexhypoxic mouse bioassay adapted from the method described by Cotes andBangham (1961), Nature 191: 1065-1067. This assay examines the abilityof a test peptide to function as an EPO mimetic: i.e., to activate EPO-Rand induce new red blood cell synthesis. Red blood cell synthesis isquantitated based upon incorporation of radiolabeled iron intohemoglobin of the synthesized red blood cells.

BDF1 mice are allowed to acclimate to ambient conditions for 7-10 days.Body weights are determined for all animals, and low weight animals (<15grams) are not used. Mice are subjected to successive conditioningcycles in a hypobaric chamber for a total of 14 days. Each 24 hour cycleconsists of 18 hr at 0.40±0.02% atmospheric pressure and 6 hr at ambientpressure. After conditioning the mice are maintained at ambient pressurefor an additional 72 hr prior to dosing.

Test peptides, or recombinant human EPO standards, are diluted inPBS+0.1% BSA vehicle (PBS/BSA). Peptide monomer stock solutions arefirst solubilized in dimethyl sulfoxide (DMSO). Negative control groupsinclude one group of mice injected with PBS/BSA alone, and one groupinjected with 1% DMSO. Each dose group contains 10 mice. Mice areinjected subcutaneously (scruff of neck) with 0.5 mL of the appropriatesample.

Forty eight hours following sample injection, the mice are administeredan intraperitoneal injection of 0.2 ml of Fe⁵⁹ (Dupont, NEN), for a doseof approximately 0.75 μCuries/mouse. Mouse body weights are determined24 hr after Fe⁵⁹ administration, and the mice are sacrificed 48 hr afterFe⁵⁹ administration. Blood is collected from each animal by cardiacpuncture and hematocrits are determined (heparin was used as theanticoagulant). Each blood sample (0.2 ml) is analyzed for Fe⁵⁹incorporation using a Packard gamma counter. Non-responder mice (i.e.,those mice with radioactive incorporation less than the negative controlgroup) are eliminated from the appropriate data set. Mice that havehematocrit values less than 53% of the negative control group are alsoeliminated.

Results are derived from sets of 10 animals for each experimental dose.The average amount of radioactivity incorporated [counts per minute(CPM)] into blood samples from each group is calculated.

2. Reticulocyte Assay

Normal BDF1 mice are dosed (0.5 mL, injected subcutaneously) on threeconsecutive days with either EPO control or test peptide. At day three,mice are also dosed (0.1 mL, injected intraperitoneally) with irondextran (100 mg/ml). At day five, mice are anesthetized with CO₂ andbled by cardiac puncture. The percent (%) reticulocytes for each bloodsample is determined by thiazole orange staining and flow cytometeranalysis (retic-count program). Hematocrits are manually determined. Thecorrected percent of reticulocytes is determined using the followingformula:

% RETIC_(CORRECTED)=%RETIC_(OBSERVED)×(Hematocrit_(INDIVIDUAL)/Hematocrit_(NORMAL))

3. Hematological Assay

Normal CD1 mice are dosed with four weekly bolus intravenous injectionsof either EPO positive control, test peptide, or vehicle. A range ofpositive control and test peptide doses, expressed as mg/kg, are testedby varying the active compound concentration in the formulation. Volumesinjected are 5 ml/kg. The vehicle control group is comprised twelveanimals, while 8 animals are in each of the remaining dose groups. Dailyviability and weekly body weights are recorded.

The dosed mice are mice are fasted and then anesthetized with inhaledisoflurane and terminal blood samples are collected via cardiac orabdominal aorta puncture on Day 1 (for vehicle control mice) and on Days15 and 29 (4 mice/group/day). The blood is transferred to Vacutainer®brand tubes. Preferred anticoagulant is ethylenediaminetetraacetic acid(EDTA).

Blood samples are evaluated for endpoints measuring red blood synthesisand physiology such as hematocrit (Hct), hemoglobin (Hgb) and totalerythrocyte count (RBC) using automated clinical analysers well known inthe art (e.g., those made by Coulter, Inc.).

Data for representative EPO-R agonist peptides in this assay are givenin Table 4. Results are given as increase in percent (%) hematocrit(Ht), relative to vehicle injected control mice, at day 15 and at day29. The indicated peptide compounds were administered to test mice at adose of 1 mg/kg.

TABLE 4 In vivo hematological assay for peptide dimers Increase IncreaseCompound in Ht (%) in Ht (%) designation Peptide dimer at day 15 at day29 AF35526

22.1 28.6 AF35527

6.8 15.9 AF35563

18.3 23.8 AF35594

7.9 12.8

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values are approximate, and areprovided for description.

Numerous references, including patents, patent applications, and variouspublications are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the presentinvention. All references cited and discussed in this specification areincorporated herein by reference in their entirety and to the sameextent as if each reference was individually incorporated by reference.

1. A method for treating a patient, comprising administering to apatient having a disorder characterized by a deficiency oferythropoietin or a low or defective red blood cell population, atherapeutically effective amount of a peptide comprising about 17 toabout 40 amino acid residues in length and comprising the amino acidsequence:LYACHX₀GPITX₁VCQPLR(SEQ ID NO:1) wherein X₀ is a residue selected fromthe group consisting of methionine (M) and homoserine methylether (Hsm),and X₁ is a residue selected from tryptophan (W), 1-naphthylalanine(1-nal), and 2-naphthylalanine (2-nal). 2-29. (canceled)